Compositions And Methods Using Split Polymerases

Attorney Docket No: STG-188 Customer No.: 27,495

COMPOSITIONS AND METHODS USING SPLIT POLYMERASES

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application relies on the disclosure of and claims the benefit of the filing dates of U.S. provisional patent application number 60/878,020, filed on 29 December

2006, and U.S. provisional patent application number 60/881,694, filed on 19 January

2007, the entire disclosures of both of which are hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[002] The present invention relates to compositions and methods utilizing split polymerases composed of at least two discrete polypeptides that stably associate to form a single polymerase. The invention further relates to nucleic acid constructs for expressing the split polymerases of the invention, and methods for making the split polymerases of the invention. The enzymes of the invention are useful in many applications calling for DNA synthesis. Description of Related Art

[003] Detectable labeling of nucleic acids is required for many applications in molecular biology, including applications for research as well as clinical diagnostic techniques. A commonly used method of labeling nucleic acids uses one or more unconventional nucleotides and a polymerase enzyme that catalyzes the template-dependent incorporation of the unconventional nucleotide(s) into the newly synthesized complementary strand.

[004] The ability of a DNA polymerase to incorporate the correct deoxynucleotide is the basis for high fidelity DNA replication in vivo. Amino acids within the active site of polymerases form a specific binding pocket that favors the placement of the correct complementary nucleotide opposite the template nucleotide. If a mismatched nucleotide, ribonucleotide, or nucleotide analog fills that position, the precise alignment of the amino acids contacting the incoming nucleotide may be distorted into a position unfavorable for Attorney Docket No: STG-188 Customer No.: 27,495

DNA polymerization. Because of this, the unconventional nucleotides or nucleotide analogs used to label DNA tend to be incorporated into the elongated strand less efficiently than do the standard deoxynucleotide triphosphates (dNTPs; the so-called "standard" dNTPs include deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and thymidine triphosphate (dTTP, also called TTP)).

[005] The reduced efficiency with which unconventional nucleotides are incorporated by the polymerase increases the amount of the unconventional nucleotide necessary for DNA labeling. The reduced efficiency of incorporation of a particular nucleotide can also adversely affect the performance of techniques or assays, such as DNA sequencing, which depend upon unbiased incorporation of unconventional nucleotides for homogeneous signal strength.

[006] The identity and exact arrangement of the amino acids of a DNA polymerase that contact an incoming nucleotide triphosphate determine the nature of the nucleotides, both conventional and unconventional, that may be incorporated by that polymerase enzyme. Changes in the exact placement of the amino acids that contact the incoming nucleotide triphosphate at any stage of binding or chain elongation can dramatically alter the polymerase's capacity for utilization of unusual or unconventional nucleotides. Sometimes changes in distant amino acids can influence the incorporation of nucleotide analogs due to indirect global or structural effects. Polymerases with increased capacity to incorporate nucleotide analogs are useful for labeling DNA or RNA strands with nucleotides modified with signal moieties such as dyes, reactive groups or unstable isotopes.

[007] In addition to labeled nucleotides, an extremely important class of modified nucleotides is the dideoxynucleotides. The so-called "Sanger" or "dideoxy" DNA sequencing method (Sanger et al, 1977, Proc. Natl. Acad. Sci. USA 74: 5463, which is incorporated herein by reference) relies upon the template-directed incorporation of nucleotides onto an annealed primer by a DNA polymerase from a mixture containing deoxy- and dideoxynucleotides. The incorporation of a dideoxynucleotide results in Attorney Docket No: STG-188 Customer No.: 27,495 chain termination, the inability of the enzyme to catalyze further extension of that strand. Electrophoretic separation of reaction products results in a "ladder" of extension products wherein each extension product ends in a particular dideoxynucleotide complementary to the nucleotide opposite it in the template. The distance of the dideoxynucleotide analog from the primer is indicated by the length of the extension product. When four reactions, each containing one of the four dideoxynucleotide analogs ddA, ddC, ddG, or ddT (ddNTPs) are separated on the same gel, the sequence of the template may be read directly from the ladder patterns. Extension products may be detected in several ways, including for example, the inclusion of isotopically- or fluorescently-labeled primers, deoxynucleotide triphosphates or dideoxynucleotide triphosphates in the reaction. [008] Fluorescent labeling has the advantages of faster data collection, since detection may be performed while the gel is running, and longer reads of sequence data from a single reaction and gel are possible. Further, fluorescent sequence detection has allowed sequencing to be performed in a single reaction tube containing four differentially- labeled fluorescent dye terminators (the so-called dye -terminator method, Lee et al., 1992, Nucleic Acids Res. 20: 2471, incorporated herein by reference). [009] A desirable quality of a polymerase useful for DNA sequencing is improved incorporation of dideoxynucleotides. Improved incorporation of dideoxynucleotides can make processes such as DNA sequencing more cost effective by reducing the requirement for expensive radioactive or fluorescent dye-labeled dideoxynucleotides. Moreover, unbiased dideoxynucleotide incorporation provides improved signal uniformity, leading to increased accuracy of base determination. The even signal output further allows subtle sequence differences caused by factors like allelic variation to be detected. Allelic variation, which produces two different half strength signals at the position of relevance, can easily be concealed by the varied signal strengths caused by polymerases with non-uniform ddNTP utilization.

[010] Dual-labeled nucleotide analogs (see, e.g., US Patent Publication 20040014096) are nucleotide analogs that have both fluorescent and quenching groups attached, resulting in a molecule that is non-fluorescent before it is incorporated, whereby the Attorney Docket No: STG-188 Customer No.: 27,495 fluorescent group is cleaved off of the nucleotide. Dual-labeled nucleotide analogs containing both a fluorescent moiety and a quencher moiety can be used as chain terminators in place of dideoxynucleotide chain terminators commonly used in the art. A chain terminating dual-labeled nucleotide analog has a sugar moiety which is, or is equivalent to a 2',3'-dideoxypyrofuranose molecule. The dual-labeled nucleotide analogs have the advantage of reduced background fluorescence compared with more traditionally labeled chain terminating nucleotide analogs. Since the dual-labeled nucleotide analogs do not emit a fluorescent signal unless they are incorporated into a polynucleotide chain, background fluorescence resulting from unincorporated analogs is significantly reduced. Dual-labeled nucleotide analogs are also useful for monitoring progress of real time amplification in quantitative PCR (QPCR) methods. [Oil] The use of such dual-labeled analogs is limited by the low utilization of such analogs by polymerases. In order to promote incorporation of the analogs into the growing strand, relatively high concentrations of the analogs must be used. The analogs are expensive and decrease the rate of extension, potentially decreasing processivity of the polymerase. High concentrations of the dual-labeled analogs can also result in increased background signal and inter-molecular quenching. A polymerase with reduced discrimination towards dual-labeled nucleotide analogs could result in decreased cost by decreasing the amount of analog required per reaction, while increasing fluorescent signal and sensitivity in both QPCR and sequencing reactions.

[012] Incorporation of ribonucleotides by the native form of DNA polymerase is a rare event. Mutants that incorporate higher levels of ribonucleotides can be used for applications such as sequencing by partial ribosubstitution. In this system, a mixture of ribonucleotides and deoxynucleotides corresponding to the same base are incorporated by the mutant polymerase (Barnes, 1978 J. MoI. Biol. 119:83-99). When the ribosequencing reactions are exposed to alkaline conditions and heat, fragmentation of the extended strand occurs. If the reactions for all four bases are separated on a denaturing acrylamide gel, they produce a sequencing ladder. The applicants of the present patent application have realized that there is a need in the art for polymerase mutants with higher utilization Attorney Docket No: STG-188 Customer No.: 27,495 of ribonucleotides for this alternative method of sequencing.

[013] Alternatively, the incorporation of ribonucleotides followed by alkaline hydrolysis can be utilized in a system that requires random cleavage of DNA molecules such as DNA shuffling ((Stemmer, 1994, Nature, 370: 389-391), which has also been called molecular breeding, sexual PCR, and directed evolution.

[014] Another desirable quality in a DNA labeling enzyme is thermal stability. DNA polymerases exhibiting thermal stability have revolutionized many aspects of molecular biology and clinical diagnostics since the development of the polymerase chain reaction (PCR), which uses cycles of thermal denaturation, primer annealing, and enzymatic primer extension to amplify DNA templates. The prototype thermostable DNA polymerase is Taq polymerase, originally isolated from the thermophilic eubacterium Thermus aquaticus. So-called "cycle sequencing" reactions using thermostable DNA polymerases have the advantage of requiring smaller amounts of starting template relative to conventional (i.e., non-cycle) sequencing reactions.

[015] There are three major families of DNA polymerases, termed families A, B, and C. The classification of a polymerase into one of these three families is based on structural similarity of a given polymerase to E. coli DNA polymerase I (Family A), II (Family B) or III (Family C). As examples, Family A DNA polymerases include, but are not limited to Klenow DNA polymerase, Thermus aquaticus DNA polymerase I (Taq polymerase) and bacteriophage T7 DNA polymerase; Family B DNA polymerases, formerly known as α -family polymerases (Braithwaite and Ito, 1991, Nuc. Acids Res. 19:4045), include, but are not limited to human α, δ and ε DNA polymerases, T4, RB69, and Φ29 bacteriophage DNA polymerases, and Pyrococcus furiosus DNA polymerase (Pfu polymerase); and family C DNA polymerases include, but are not limited to Bacillus subtilis DNA polymerase III, and E. coli DNA polymerase III α and ε subunits (listed as products of the dnaE and dnaQ genes, respectively, by Braithwaite and Ito, 1993, Nucleic Acids Res. 21: 787). An alignment of DNA polymerase protein sequences of each family across a broad spectrum of archaeal, bacterial, viral, and eukaryotic organisms is presented in Braithwaite and Ito (1993, supra), which is incorporated herein by reference. Attorney Docket No: STG-188 Customer No.: 27,495

[016] As shown in Braithwaite and Ito (1993, supra), within regions I, II, and III, a set of highly conserved residues form three chemically distinct clusters consisting of exposed, aromatic residues (RB69 numbering, Y416, Y567, and Y391), negatively charged residues (D621, D623, D41 1, D684, and E686), and a positively charged cluster (K560, R481, and K486). Comparison with a Taq polymerase-DNA complex suggests that these three clusters encompass the region in which the primer terminus and the incoming dNTP would be expected to bind. Modeling of the dNTP and primer template complex in RB69 was carried out using the atomic coordinates of the reverse transcriptase c-DNA co-crystal. The model predicts the RB69 Y416 packs under the deoxyribose portion of the dNTP. Tyrosine at this position has been implicated in ribose selectivity, contributing to polymerase discrimination between ribonucleotides and deoxyribonucleotides in mammalian reverse transcriptases (Yl 15) (Gao et al., 1997, Proc. Natl. Acad. Sci. USA 94:407; Joyce, 1994, Proc. Natl. Acad. Sci. USA 94:1619). [017] Region III of the Family B polymerases (also referred to as motif B) has also been demonstrated to play a role in nucleotide recognition. This region, which corresponds to AA 487 to 495 of JDF-3 Family B DNA polymerase, has a consensus sequence KX3 NSXYG (SEQ ID NO:1) (Jung et al., 1990, supra; Blasco et al., 1992, supra; Dong et al., 1993, J. Biol. Chem. 268:21163; Zhu et al., 1994, Biochem. Biophys. Acta 1219:260; Dong and Wang, 1995, J. Biol. Chem. 270:2 1563), and is functionally, but not structurally (Wang et al., 1997, supra), analogous to KX3 (FA^)GX2YG (SEQ ID NO: 2) in helix O of the Family A DNA polymerases. In Family A DNA polymerases, such as the fragment and Taq DNA polymerases, the O helix contains amino acids that play a major role in dNTP binding (Astatke al., 1998, J. MoI. Biol. 278:147; Astatke et al., 1995, J. Biol. Chem. 270:1945; Polesky et al., 1992, I. Biol. Chem 267:8417; Polesky et al., 1990, J. Biol. Chem. 265:14579; Pandey et al., 1994, J. Biol. Chem. 269:13259; Kaushik et al., 1996, Biochem. 35:7256). Specifically, helix O contains the F (F763 in the fragment; F667 in Taq) which confers ddNTP discrimination in Family A DNA polymerases (KX3(FA^)GX2YG; SEQ ID NO: 2) (Tabor and Richardson, 1995, supra). [018] The term used to describe the tendency of DNA polymerases to not incorporate Attorney Docket No: STG-188 Customer No.: 27,495 unnatural nucleotides into the nascent DNA polymer is "discrimination". In Family A DNA polymerases, the effective discrimination against incorporation of dideoxynucleotide analogs is largely associated with a single amino acid residue. The majority of enzymes from the Family A DNA polymerases have a phenylalanine (phe or F) residue at the position equivalent to F762 in E. coli fragment of DNA polymerase and demonstrate a strong discrimination against dideoxynucleotides. A few polymerases (e.g. T7 DNA polymerase) have a tyrosine (tyr or Y) residue at the corresponding position and exhibit relatively weak discrimination against dideoxynucleotides. Family A polymerases with tyrosine at this position readily incorporate dideoxynucleotides at levels equal to or only slightly different from the levels at which they incorporate deoxynucleotides. Conversion of the tyrosine or phenylalanine residues in the site responsible for discrimination reverses the dideoxynucleotide discrimination profile of the Family A enzymes (Tabor and Richardson, 1995, Proc. Natl. Acad. Sci. USA 92:6449).

[019] Among the thermostable DNA polymerases, a mutant form of the Family A DNA polymerase from Thermus aquaticus, known as AmpliTaq FS® (Perkin Elmer), contains a F667Y mutation at the position equivalent to F762 of DNA polymerase and exhibits increased dideoxynucleotide uptake (i.e., reduced discrimination against ddNTPs) relative to the wild-type enzyme. The reduced discrimination for dideoxynucleotide uptake makes it more useful for fluorescent and labeled dideoxynucleotide sequencing than the wild-type enzyme.

[020] The F667Y mutant of Taq DNA polymerase is not suited for use with fluorescein- labeled dideoxynucleotides, necessitating the use of rhodamine dye terminators. Rhodamine dye terminators that are currently utilized with Taq sequencing reactions stabilize DNA secondary structure, causing compression of signal. Efforts to eliminate compression problems have resulted in systems that use high amounts of the nucleotide analog deoxyinosine triphosphate (dITP) in place of deoxyguanosine triphosphate. While incorporation of (dITP) reduces the compression of the signal, the presence of dITP in the reaction produces additional complications including lowered reaction temperatures and Attorney Docket No: STG-188 Customer No.: 27,495 increased reaction times. Additionally, the use of rhodamine dyes in sequencing requires undesirable post-reaction purification (Brandis, 1999 Nuc. Acid Res. 27:1912). In the Family A E. coli DNA polymerase I fragment, modification of a conserved glutamate residue (E7 10) reduces discrimination against ribonucleotides (Astatke et al., 1998, Proc. Natl. Acad. Sci. USA 96:3402).

[021] In Family A DNA polymerases, such as the Klenow fragment and Taq DNA polymerases, the O helix contains amino acids that play a major role in dNTP binding (Astatke et al., 1998, J. MoI. Biol. 278:147; Astatke et al., 1995,1 Biol. Chem. 270:1945; Polesky et al., 1992, J. Biol. Chem 267:84 17; Polesky et al., 1990, J. Biol. Chem.265:U579; Pandey et al., 1994, J. Biol. Chem. 269:13259; Kaushik et al., 1996, Biochem. 35:7256). Specifically, helix O contains the F (F763 in the Klenow fragment; F667 in Taq) which confers ddNTP discrimination in Family A DNA polymerases (KX3(FA^)GX2YG; SEQ ID NO: 2) (Tabor and Richardson, 1995, supra). [022] With the exception of the position of acidic residues involved in catalysis in the so-called palm domain, Family B DNA polymerases exhibit substantially different structure compared to Family A DNA polymerases (Wang et al., 1997, Cell 89:1087; Hopfner et al., 1999, Proc. Natl. Acad. Sci. USA 96:3600). The unique structure of Family B DNA polymerases may permit a completely different spectrum of interactions with nucleotide analogs, perhaps allowing utilization of analogs that are unsuitable for use with Family A DNA polymerases due to structural constraints. Thermostable Family B DNA polymerases have been identified in hyperthermophilic archaea. These organisms grow at temperatures higher than 910C and their enzymes demonstrate greater thermostability (Mathur et al., 1992, Stratagies 5: 11) than the thermophilic eubacterial Family A DNA polymerases. Alignments of a number of Family B DNA polymerases can be seen in Figures 2 and 6.

[023] Structural analysis of A family polymerases, Pol β, HIV reverse transcriptase, and the B family polymerase gp43 demonstrate that all share a functional polymerase structure which resembles a right hand built by the palm, fingers, and thumb domains (see Brautigman and Steitz, 1998, Curr Opin Struc Biol 8:54 for review, incorporated Attorney Docket No: STG-188 Customer No.: 27,495 herein by reference). The palm domains show a similar topology among all families, except Pol β. The fingers and thumb domain are highly diverse among the different families, and although the thumb domains are mainly alpha-helical, the detailed structures of the domains are not related. Perhaps surprisingly, the fingers and thumb domains in all four families have arisen from different ancestors. [024] As polymerases are used for many laboratory applications, a number of polymerases have been developed to have properties that are desirable for a variety of laboratory applications. For example, mutations at sites corresponding to amino acids E 141 and D 143 in Pyrococcus furiosus (Pfu) (SEQ ID NO: 3) are known to eliminate 3 'to 5' exonuclease activity. Mutations at sites corresponding to amino acids L409, Y410, P411, R461, K465, Q472, A486, R488, L490, A491, N492, Y495, and Y497are known to reduce nucleotide discrimination in polymerases (see, e.g., U.S. Patent No. 6,946,273, U.S. Patent No. 6,333,183, U.S. Patent No. 5,882,904, U.S. Patent No. 5,827,716, Yang et al. 1999 Biochemistry 38:8094, Gardner and Jack, 1999 Nucleic Acids Research 27:2545, incorporated herein by reference). A mutation at amino acid V93, specifically V93R, (Pfu numbering) is known to disrupt uracil detection. A non-sequence-specific DNA binding domain, such as the DNA binding domain of Sso7d, can be incorporated into a polymerase to increase the processivity of the polymerase. Moreover, sites corresponding to the amino acids provided in Pfu DNA polymerase can be easily mapped onto other Family B polymerase sequences using published sequence alignments {e.g. , Braithwaite and Ito, 1993, supra; Brautigman and Steitz, 1998, supra; and Hopfner et al., 1999, supra; Biles and Connolly, 2004, supra; Gardner and Jack, 1999, supra; Edgell et al., 1997. J. Bacteriol. 179:2632) or any of a number of sequence alignment programs such as BLAST).

[025] Introducing splits into enzymes as a strategy to broaden substrate utilization is very different from currently used approaches, which are based on amino acid replacements. There are four examples of natural splits in the polymerase family. The T4-phage family includes five members that contain splits within the fingers domain (Petrov et al (2006) J MoI Biol. 361 :46-68). These splits occur naturally and it is Attorney Docket No: STG-188 Customer No.: 27,495 unknown whether the split enzymes exhibit unique characteristics such as broader substrate utilization compared to non-split T4-like phage DNA polymerases. The second natural split is the one reported in the archaeal Methanobacterium thermoautotrophicum DNA polymerase (Kelman et al (99) JBC 274:28751-61). This split also occurs naturally and is found downstream (outside) of the fingers domain. This split has also not been characterized in terms of whether it exhibits broader substrate utilization compared to non-split archaeal DNA polymerases. In the two examples of natural splits, the polymerase fragments are encoded by distinct genes that are separated by anywhere from 2bp to 3kb (T4-like phage) to 85Kbp (Mth) in the genome. The third example of a natural split is in the archaeal DNA polymerase gene. However, this split occurs within a mini-intein of N. equitans DNA polymerase, where the polymerase is expressed as two separate polypeptides, which are then spliced together (trans-splicing) to create a full length polymerase. The split is located outside of the fingers domain and has additional sequence (inteins) to stabilize the protein until the splicing event is complete (Choi et al. (06) J. MoI. Biol. 356:1093-1106.) The fourth example of a natural split is found in the archael Sulfolobus solfataricus DNA polymerase Bl (Savino et al. (2004) Structure. 12:2001-2008). In this case, the polymerase is proteolytically cleaved to produce two active fragments, a 50 kD fragment with DNA polymerase activity and a 40 kD fragment with exonuclease activity. However, the authors do not state whether these activities are reduced relative to wild type nor have the proteolytic fragments been tested for alternative or improved activities. The split in this example is also found outside the fingers domain.

[026] Polymerases having reduced discrimination are useful for applications that require incorporation of non-conventional nucleic acids. Such applications include the labeling of nucleic acid arrays, often referred to as nucleic acid or DNA "chips", in the simultaneous analyses of multiple different nucleic acid sequences. Many of these applications, such as those described in U.S. Patent No. 5,882,904 (Riedl et al.), will benefit from DNA polymerases exhibiting reduced discrimination against the incorporation of non-conventional nucleotides, particularly fluorescently-labeled non- Attorney Docket No: STG-188 Customer No.: 27,495 conventional nucleotides. Applications being addressed in the chip format include DNA sequencing and mutation detection, among others. Examples include the "mini- sequencing" methods (e.g., Pastinen et al., 1997, Genome Res. 7: 606; Syvanen, 1999, Human Mutation 13: 1-10) and the arrayed primer extension (APEX) mutation detection method (Shumaker et al., 1996, Hum. Mutat. 7: 346).

[027] The present applicants have recognized that there is a need in the art for a non- discriminating DNA polymerase for use in chip or gel based mini-sequencing systems. Such a system would advantageously permit detection of multiplexed single nucleotide polymorphisms (SNPs) and allow for quantitative genotyping. Identification of sequence variation permits the diagnosis and treatment of genetic disorders, predisposition to multifactorial diseases, and sensitivity to new or existing pharmaceutical products. [028] Additionally, the applicants have recognized that there is a need in the art for DNA polymerases with reduced discrimination against unconventional nucleotides. They have realized that there is particularly a need in the art for thermostable DNA polymerases exhibiting reduced discrimination against dideoxynucleotides, and further, for DNA polymerases exhibiting reduced discrimination against fluorescently labeled dideoxynucleotides. They have also recognized that there is a particular need for thermostable DNA polymerases exhibiting reduced discrimination against nucleotide analogs containing modifications in the polyphosphate portion of a nucleotide, especially dual-labeled oligonucleotides.

SUMMARY OF THE INVENTION

[029] The present invention relates to enzymes into which one or more non-naturally occurring splits have been introduced to broaden substrate utilization of the enzyme. The invention can be applied to many different types of enzymes. Without limiting the invention, such a non-natural split is expected to increase flexibility between sub- domains and reduce constraints on substrate interactions. In the class of enzymes called polymerases, a split in the polymerase has been found to modify activity of the polymerase. Specifically, enzymes of this class have reduced discrimination, especially Attorney Docket No: STG-188 Customer No.: 27,495 for dual-labeled nucleotide analogs, which makes them useful for applications such as QPCR and nucleic acid sequencing.

[030] In one aspect, the present invention provides non-natural polymerases containing at least one split, and compositions comprising such polymerases. These include, but are not limited to, reverse transcriptase, RNA polymerase, DNA polymerase, and DNA primase. In one embodiment, the present invention provides split polymerases from the A Family polymerases, such as the Family A polymerase from Thermus aquaticus. In another embodiment, the present invention includes split polymerases from the archaeal B Family polymerases, such as Thermococcus litoralis (Vent) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Thermococcus JDF-3 DNA polymerase, Pyrococcus horikoshii (Pho) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Pyrococcus sp. GBD (Deep Vent) DNA polymerase, Sulfolobus solfataricus DNA polymerase, and archaeal polymerase I. In still another embodiment, the split polymerase is a chimeric polymerase, a combination of an N-terminal fragment from a first polymerase, and a C-terminal fragment of a second polymerase. In a preferred embodiment, the fragments are from the same family of polymerases {e.g., both Family B polymerases).

[031] The split polymerases may be comprised of other modifications or mutations in the coding sequence, either at the region of the split and/or at regions distant from where the split occurs to further improve non-natural substrate utilization, to improve stability of the split polymerase, to remove 3 '-5' exonuclease activity, to eliminate uracil detection, to increase processivity, etc.. Other additions to the coding sequence may allow insertion of proteolytic cleavage sites to make a post-translational split of the polymerase, insertion of "slippery sequences" that viruses use to cause frameshifts that can result in a translational start and stop (process is called programmed ribosomal frameshifting; Hansen et al, 2007, PNAS 104 (14):5830-5835), or may allow other ways of making a split polymerase. The polymerases can be provided as isolated or purified substances, or as part of compositions that include one or more additional substances (e.g., water or an aqueous solution). As used herein, the terms "isolated" and "purified" Attorney Docket No: STG-188 Customer No.: 27,495 are used interchangeably to indicate that the subject protein, nucleic acid, etc. has been removed from its natural environment and preferably has been removed from the presence of one, some, substantially all, or all of the substances that are found to be present in the natural environment of the protein, nucleic acid, etc. [032] In another aspect, the invention relates to nucleic acids encoding split polymerases of the invention. A split polymerase is understood to be a polymerase expressed as at least two discrete polypeptides (e.g., an N-terminal fragment and a C- terminal fragment) that associate non-covalently to form a single, functional polymerase molecule. In a preferred embodiment, the split polymerases of the invention are encoded by a polycistronic operon in which expression of each portion or cistron of the split polymerase is translationally coupled to translation of at least one other portion of the split polymerase. In one embodiment, the polycistronic coding sequence contains at least one ribosomal reinitiation signal proximal to a frameshift termination codon. In an alternative embodiment, the split polymerases of the invention are encoded by two coding sequences that are present on two separate plasmids in the same host cell, or in two separate host cells. The nucleic acids can be provided as purified or isolated substances, alone or in compositions comprising at least one other substance (e.g., water or an aqueous solution).

[033] In an additional aspect, the invention provides methods of making a split polymerase of the invention. In general, the method comprises introducing into a host cell a nucleic acid encoding a split polymerase wherein the coding region is operably linked to a promoter, and culturing the cell under conditions that permit production of the split polymerase. In a preferred embodiment, the split polymerase is encoded by a polycistronic operon in which expression of each portion or cistron of the split polymerase is translationally coupled to translation of at least one other portion of the split polymerase. Stated another way, the method comprises using a polycistronic coding sequence operably linked to a promoter sequence functional in a host cell into which the coding sequence is inserted. In an alternative embodiment, the split polymerase of the invention is encoded by two coding sequences that are present on two separate plasmids Attorney Docket No: STG-188 Customer No.: 27,495 in the same host cell, or in two separate host cells. In both embodiments, each segment of the split polymerase is translated and the segments then assemble together, either in vivo or in vitro, to form the complete split polymerase.

[034] In yet a further aspect, the invention provides methods of using the split polymerase of the invention. A split polymerase of the invention can be used for nucleic acid amplification, nucleic acid sequencing, quantitative PCR (QPCR), nucleic acid labeling, for synthesis with modified primers or templates, or in any reaction that requires a polymerase. In general, these embodiments are particularly suited for the use of non- conventional nucleotides and nucleotide analogs such as dual-labeled nucleotide analogs. [035] In embodiments, the methods of the invention are used to increase utilization {e.g., binding and/or incorporation into a growing nucleic acid chain) of non-natural nucleotides. This can be accomplished by splitting a polymerase in such a way that the polymerase shows increased activity for non-natural nucleotides. Higher nucleotide analog uptake can also be increased by combining the split with other mutations in the amino acid sequence compared to using the split or the amino acid replacement alone. [036] In another aspect, the present invention provides compositions and kits comprising at least one split polymerase of the invention. Compositions may comprise at least one molecule of split polymerase of the invention along with one or more substances, which are typically substances which aid in the methods of the invention. In general, a kit according to the invention contains a sufficient amount of split polymerase to allow at least one method of using the split polymerase to occur and is comprised of at least one container. Kits generally comprise a polymerase or nucleic acid of the invention and optionally one or more other components useful for practicing a method of the invention, where the components of the kits are contained in one or more containers packaged in combination for ease of storage and use. Multiple containers are often present to allow for practice of methods of the invention multiple times. Attorney Docket No: STG-188 Customer No.: 27,495

BRIEF DESCRIPTION OF THE DRAWINGS

[037] The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the invention and, together with the written description, serve to explain various principles of the invention. It is to be understood that the drawings are not to be construed as a limitation on the scope or content of the invention. [038] Figure 1 shows a CLUSTAL W multiple sequence alignment of a series of finger domains from DNA Family B polymerases.

[039] Figures 2A-2B show an alignment of the amino acid sequences of a number of family B DNA polymerases. A. from Pyrococcus furiosus (Pfu) (SEQ ID NO: 3); Deep Vent Pyrococcus sp (SEQ ID NO: 7); Pyrococcus horikoshii (Pho) (SEQ ID NO: 8); Thermococcus gorgonarius (Tgo) (SEQ ID NO: 9); Thermococcus kodakarensis (Archaeal) (SEQ ID NO: 10) (known as Archaeal DNA polymerase I); JDF-3 Thermococcus sp.(SEQ ID NO: 11); 9°N Thermococcus sp. (SEQ ID NO: 12); and Vent Thermococcus litoralis (SEQ ID NO: 13). B. The consensus amino acids of the fingers motif SEQ ID NOS: 4, 5, and 6 are indicated.

[040] Figure 3A- 3E show the nucleic acid coding sequence and the corresponding translation product for A. Native Pfu (SEQ ID NO: 3) and B. Pfu with a seamless split after amino acid K467. The ribosomal reinitiation and frameshift termination codons are noted. Figures 3C-3E show three splits after amino acid 466 with a methionine residue inserted after the split to promote translation reinitiation. In Figure 3D, a methionine was mutated to provide a single reinitiation site.

[041] Figure 4 shows the amino acid coding sequence for the split JdF-3 DNA polymerase, JdF-3 202. Panel A depicts the amino fragment of the split (Fragment I), Panel B shows the shift from the translational reading frame of 1 to 3 when the ribosome encounters the stop codon (TGA) and reinitiates on the ATG codon, and Panel C depicts the carboxy fragment of the split (Fragment II).

[042] Figure 5 shows the amino acid coding sequence for JdF-3 Z2, a mutant with a split after amino acid 469 that results in a 10 amino acid insertion at the C-terminus of the Attorney Docket No: STG-188 Customer No.: 27,495

N-terminal fragment. Panel A depicts the amino fragment of the split, Panel B shows the shift from the translational reading frame of 1 to 3 when the ribosome encounters the stop codon (TAG) and reinitiates on the ATG codon, and Panel C depicts the carboxy fragment of the split.

[043] Figure 6 shows an alignment of amino acid sequences a number of family B DNA polymerases from Pyrococcus horikoshii (Pho) (SEQ ID NO:62) (Accession: NP_143776.1 GI: 14591688); DNA polymerase 1 Pyrococcus abyssi (Pab polymerase) (SEQ ID NO: 60) (Accession: P77916 GL6648073); DNA-dependent DNA polymerase; endonuclease [Pyrococcus sp.] (SEQ ID NO: 61) (Accession: CAA90887.1 GI: 1495770); Vent Thermococcus litoralis (SEQ ID NO: 57) (Accession: P303 17 GL232020) ; Deep Vent Pyrococcus sp (SEQ ID NO: 64) (Accession: Q51334 GL2494186); 9°N Thermococcus sp. (90N-7) (SEQ ID NO: 67) (Accession: IQHTA GL8569297); Thermococcus gorgonarius (Tgo) (SEQ ID NO: 69) (Accession: ITGO A GL4699806); Thermococcus aggregans (Tfu) (SEQ ID NO: 59) (Accession: 033845 GL3913524); Pyrococcus sp. ST700 (ST700) (SEQ ID NO: 63) (Accession No. CAC 12847.1 GL10799869); Pyrococcus furiosus (Pfu) (SEQ ID NO: 66) (Accession: AAL8 0336.1 01 :18892147); DNA polymerase and endonucleases Thermococcus sp.GES (SEQ ID NO: 68) (Accession: CAC12850.1 GL10799895); Thermococcus litoralis DNA dependent DNA polymerase (TLI) (SEQ ID NO: 58; (Accession: AAA721O1.1 GL348689); DNA-dependent polymerase precursor Thermococcus sp. NAl (TSPNAl) (SEQ ID NO: 71) (Accession No: ABCl 1972.1 GL83338486); DNA-directed DNA polymerase (EC 2.7.7.7) KOD, intein containing precursor Pyrococcus sp. (strain KODl) (TSPKOD) (SEQ ID NO: 72) (Accession: ABCl 1972.1 GL83338486); PoIA precursor Thermococcus zilligii (TZI)(SEQ ID NO: 70)(Accession: ABD 14868.1 GL86753389); Thermococcus kodakarensis (THY) (SEQ ID NO: 73) (Accession: 1WN7_A GL73535447); Pyrococcus glycovorans (PGL) (SEQ ID NO: 65) (Accession: CAC 12849.1 GL10799899); DNA polymerase II Sulfolobus tokodai str. 7 (SULFOTO) (SEQ ID NO: 74) (Accession: NP378066.1 GI: 15922397); and Desulfurococcus sp. Tok (TOK) (SEQ ID NO: 15) (Accession: 1D5AA GL7546394). [044] Figure 7 shows an amino acid alignment of the finger domain of a number of Attorney Docket No: STG-188 Customer No.: 27,495 family B DNA polymerases from JDF-3 Thermococcus sp. (SEQ ID NO: 33); Pyrococcus horikoshii (Pho) (SEQ ID NO:34) (Accession: NPJ43776.1 GI: 14591688); DNA polymerase 1 Pyrococcus abyssi (Pab polymerase) (SEQ ID NO: 35) (Accession: P779 16 01 :6648073); DNA-dependent DNA polymerase; endonuclease [Pyrococcus sp.} (SEQ ID NO: 36) (Accession: CAA90887.1 GI: 1495770); Vent Thermococcus litoralis (SEQ ID NO: 37) (Accession: P303 17 GL232020) ; Deep Vent Pyrococcus sp (SEQ ID NO: 38) (Accession: Q51334 GL2494186); 9°N Thermococcus sp. (9ON-7) (SEQ ID NO: 39) (Accession: IQHTA GL8569297); Thermococcus gorgonarius (Tgo) (SEQ ID NO: 40) (Accession: ITGO A GL4699806); Thermococcus aggregans (Tfu) (SEQ ID NO: 41) (Accession: 033845 GI:39 13524); Pyrococcus sp. ST700 (ST700) (SEQ ID NO: 42) (Accession No. CAC12847.1 01 :10799869); Pyrococcus furiosus (Pfu) (SEQ ID NO: 43) (Accession: AAL80336.1 01 :18892 147); DNA polymerase and endonucleases Thermococcus sp. GE8 (SEQ ID NO: 44) (Accession: CAC 12850.1 01 :10799895); Thermococcus litoralis DNA dependent DNA polymerase (TLI) (SEQ ID NO: 45) (Accession: AAA72 101.1 GL348689); DNA-dependent polymerase precursor hermococcus sp. NAl (TSPNAl) (SEQ ID NO: 46) (Accession No: ABCI 1972.1 01 :83338486); DNA-directed DNA polymerase (EC 2.7.7.7) KOD, intein containing precursor - Pyrococcus sp. (strain KODl) (TSPKOD) (SEQ ID NO: 47) (Accession: ABCl 1972.1 01 :83338486); PoIA precursor Thermococcus zilligii (TZI)(SEQ ID NO: 48)(Accession: ABD14868.1 GL86753389); Thermococcus kodakarensis (THY) (SEQ ID NO: 49) (Accession: 1WN7_A GL73535447); Pyrococcus glycovorans (PGL) (SEQ ID NO: 50) (Accession: CAC12849.1 01 :10799899); DNA polymerase II Sulfolobus tokodaz str. 7 (SULFOTO) (SEQ ID NO: 51) (Accession: NP 378066.1 GL15922397); and Desulfurococcus sp. Tok (TOK) (SEQ ID NO: 52) (Accession: 1D5AA GL7546394). The consensus amino acids of the fingers motif SEQ ID NOS: 53 and 54 including DNA polymerase II Sulfolobus tokodai str. 7, and SEQ ID NOS: 55 and 56 excluding DNA polymerase II Sulfolobus tokodai str. 7 are indicated. In SEQ ID NOs: 54 and 56, B is M, V, L, or I; J is S or T; 0 is D or E; U is F or Y; Z is Q, K, or R; and X is any amino acid. Attorney Docket No: STG-188 Customer No.: 27,495

[045] Figure 8 depicts PCR amplification activity of several of the JdF-3 variants of the invention.

[046] Figure 9 shows the amino acid sequence of the amino fragment (Panel A) and the carboxyl fragment (Panel B) of the 4Cl 1 DNA polymerase with an additional Q484R mutation.

[047] Figure 10 shows PCR amplification activity of the frameshift (split) mutant with an additional Q484R mutation (double mutant) in the presence of a dCTP analog labeled with dabcyl at the gamma phosphate.

[048] Figure 11 depicts PCR amplification activity of the 4Cl 1 mutant with the exo minus mutations in the presence of dCTP labeled with the nucleotide analog FCD. In panel A, the gel is unstained and the PCR products are revealed by the fluorescent label in the nucleotide. In panel B the gel is stained with ethidium bromide to show total

DNA. The micromolar concentration of analog is shown above the well.

[049] Figure 12 shows SDS-PAGE analysis of solubilized, separately expressed 4Cl 1 split polymerase fragments after partial purification from inclusion bodies (Panel A and

B), SDS-PAGE analysis of refolded 4Cl 1 split polymerase (Panel C), PCR assays of the refolded protein (Panel D) and SDS-PAGE analysis of the refolded protein after further purification by column chromatography (Panel E).

[050] Figure 13 shows the amino acid sequence of the amino fragment (Panel A;

Fragment I), the shift from the translational reading frame of 1 to 3 when the ribosome encounters the stop codon (TGA) and reinitiates on the ATG codon (Panel B), and the carboxyl fragment (Panel C; Fragment II) of clone SQ, a seamless split Pfu polymerase with an additional Q484R mutation.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

[051] The ability to make mutations that alter protein structure has been a fundamental tool for elucidating protein function and harnessing proteins to complete reactions that they were previously unsuited for (Arnold, 1993, FASEB J. 7:744). Changes to alter Attorney Docket No: STG-188 Customer No.: 27,495 enzyme characteristics such as rate, thermostability, pH optimum, substrate and end products in reactions are widely used in research and industry. These changes involve changing, adding, or deleting the amino acids of the targeted enzyme. A typically undesirable mutation in these schemes is a frameshift mutation in which the addition or deletion within the nucleotide sequence causes the ribosomal translation complex to read the triplet DNA codons out of register, leading to incorrect amino acid incorporation and early truncation. In most cases, early termination of translation leads to a non- functional or poorly functioning enzyme. As reported herein, the ability to strategically cause breaks in the DNA polymerase enzyme (through start and stop codons) not only maintained polymerase function but also gave the surprising ability to accept a greater range of substrates, perhaps through increased flexibility or repositioning of critical domains

[052] The present invention relates to introducing non-natural splits into enzymes to broaden substrate utilization. By "non-natural", it is meant that the split is introduced into a site where an enzyme is not known to split naturally. It thus does not address known, naturally split polymerases, for example, but does encompass such enzymes with additional or alternative split sites. The invention can be applied to many different types of enzymes. Without limiting the invention, such a non-natural split is expected to increase flexibility between sub-domains and reduce constraints on substrate interactions. One can envision that insertions at the split site (and a corresponding removal of the split) can have a similar effect to the split, by, for example, increasing flexibility between sub- domains. One can also envision that instead of making a non-natural split polymerase, a non-natural non-split polymerase may be formed. In this embodiment, a stop codon can be removed from between a pair of tandemly encoded heterodimeric enzymes to make a monomeric enzyme. For example, the Family D Pfu DNA polymerase naturally has the configuration of two tandemly encoded heterodimeric enzymes.

[053] When the invention is used to split DNA polymerases, improved incorporation of non-natural substrates, such as dual label nucleotides (e.g., analogs as described in U.S. Patent Publication 20040014096), is seen. Splits can be introduced into the fingers Attorney Docket No: STG-188 Customer No.: 27,495 domain, the 3 ' primer binding/editing site, duplex DNA binding site, single-stranded DNA template binding domain, etc. to improve utilization of modified primed-templates or to modify other activities of a polymerase. Also, splits near the domains involved in accessory protein binding could alter interaction with these factors, leading to differences in the assembly of replication complexes and altered activity. Accessory factors for polymerases include, but not limited to, clamp (PCNA), single stranded binding proteins (RPA) and helicases.

[054] The invention comprises introducing splits in regions of the polymerase that interact with the incoming nucleotide, the 3' end of the primer, and/or the DNA template, or any other region that will improve some aspect of DNA polymerization, such as utilization of non-natural substrates.

[055] One of the split polymerases of the invention, Pfu 4Cl 1, is comprised of several mutations (1-467 V93R/A318T)/(468-775 Q484R/V604L/A662V-Ss07d7m). The nucleotide sequence that encodes Pfu 4Cl 1 (see Figure 3B, Figure 9) contains a frameshift deletion that results in premature termination of translation, as compared to the wild-type sequence, and the re-initiation of translation after a one nucleotide upstream ribosomal shift without any amino acid loss or change. This process is known as translational coupling and is found in some polycistronic operons that link the translation of a second or even third gene to the regulation and transcription of the first (Aksoy et al., 1984, J. Bacteriol. 157:363). Experimentation in E. coli has demonstrated that if translation is prematurely terminated, a methionine codon (AUG) within seven codons downstream can reinitiate translation without ribosome recycling factor or a Shine- Dalgarno consensus (Karamyshev, 2004, Biochimie.l2:933). The optimal translational coupling positions of the initiating and terminating codons have been documented as AUGA with the AUG start occurring before the UGA termination codon (Hopfner, 1999, supra). In the Pfu sequence, deletion of any single dA from nucleotides 1398 to 1402 results in the same frameshift/coupled translation end product. The truncated polypeptide (fragment I) corresponds to amino acids 1-467, while the new polypeptide corresponds to the former carboxy end of Pfu, beginning at Met 468 and encompassing amino acids 468- Attorney Docket No: STG-188 Customer No.: 27,495

755 (fragment II).

[056] Expression of the Pfu 1-467 and 468-775 fragments (produced by the frameshift), separately and together, has demonstrated that the carboxy protein domain beginning at amino acid 468 and encompassing amino acids 468-755 of SEQ ID NO: 3 is extremely insoluble when expressed apart from the amino fragment. When expressed together through coupled translation, the two peptides co-purify in equimolar amounts indicating that the fragments fold together in a stable complex. When the gene fragments encoding the two peptides are expressed in isolation from each other, the amino protein can be purified, although it does not appear to be as stable or soluble as the full-length native protein.

[057] When the Q484R mutation is added to the split, there is a synergistic effect on nucleotide analog incorporation. The two mutations are in close proximity on opposite alpha helixes near the tip of the finger. In Pfu, the split-inducing frameshift and Q484R mutations separately improve FAM-dCTP-Dabcyl uptake, while the combination of these two mutations produces even greater analog incorporation. The double mutant configuration particularly improves the uptake of dabcyl-dCTP modified at the gamma phosphate (Fig. 10). While not wishing to be bound by mechanism, it is proposed that incorporation of the bulky nucleotide analog is improved by loosening the nucleotide binding pocket when the finger folds over the incoming nucleotide. [058] The 4C 11 split Pfu and Q484R double mutant are robust in PCR reactions and demonstrate no observable defect in activity. This is somewhat surprising in view of the fact that even small changes (especially in such a highly conserved domain) can be expected to be deleterious to activity.

[059] Splits were inserted into a wild-type and a mutant Q484R Pfu polymerase background, and were tested for the ability to incorporate the bulky nucleotide analog 5- aminoallyl-(5-FAM)-2'-deoxycytidine-5'-gamma-triphospho-N6-(6-aminohexyl)-dabcyl (FCD) in an end point PCR assay. The reaction was run with a standard concentration of dATP, dGTP, and dTTP. The dCTP pool was a combination of dCTP and FCD, with the percentage of FCD in the dCTP pool varying from zero (100% dCTP:0% FCD) to one Attorney Docket No: STG-188 Customer No.: 27,495 hundred percent (0% dCTP:100% FCD). Electrophoresis of the PCR products on an agarose or acrylamide gel in the absence of ethidium bromide allowed the resultant FAM-labeled DNA products to be visualized with ultraviolet excitation and a green filter (Fig. 11). The gel was subsequently stained with ethidum bromide and visualized with an orange filter to reveal all DNA molecules. The wild type exo-minus Pfu was unable to incorporate the FCD analog, while the split Pfu Q484R double mutant showed an optimal uptake of FAM-dCMP at 50-75% FCD (under these conditions). In the present invention, "exo-minus" is defined as a polymerase comprising a D141A/E143A mutation (see, e.g., U.S. Patent No. 5,489,523).

[060] The split polymerases further containing a Q484R mutation were also tested in an exo-plus or exo-minus (not comprised of the DI41A/E143A mutation and comprised of the D141A/E143A mutation, respectively) version of Pfu DNA polymerase. Because labeled analogs can be excised by an active 3' to 5' exonuclease, amplicons generated with exo-plus enzyme are less fluorescent than amplicons generated with exo-minus split Pfu Q484R, and free FAM-dCMP released by proofreading activity is readily visible on the gel. Therefore, split polymerases lacking 3' to 5' exo activity are preferred for use for incorporation of non-conventional nucleotides in an endpoint reaction. In other applications, use of polymerases with exo activity might be preferred. [061] Coding sequence for a mutant Sso7d protein domain (<90% identity; see U.S. Patent Publication 20050048530) was incorporated at the C-terminal end of the Pfu 468- 775 (Q484R) fragment to increase the processivity of the polymerase. The polymerase was expressed from a bicistronic DNA polymerase with either the exo-plus or exo-minus version of Pfu with a split after amino acid 466. Incorporation of the DNA binding domain did not alter the discrimination properties of the polymerase as compared to a polymerase lacking a DNA binding domain.

[062] To determine if increased utilization of dual labeled nucleotide analogs by insertion of a split into a polymerase was unique to Pfu, a JDF-3 split polymerase bicistronic coding sequence was made. The resulting polymerase is shown in Figure 5 (JdF-3 Z2). In addition to the split after amino acid 469, 10 amino acids were added on Attorney Docket No: STG-188 Customer No.: 27,495 to the C-terminus of the N-terminal JdF-3 fragment. This is different from the Pfu 4Cl 1 mutation, where no amino acids were added to the wild-type sequence. The JdF-3 mutant Z2 shows an insertion in the finger near the tip of the finger domain and was found to result in increased incorporation of non-conventional nucleotides in extension reactions. [063] As mentioned above, the present invention provides enzymes with non-natural splits that broaden substrate utilization. The invention can be applied to many different types of enzymes. For example, the non-natural split can be in any region that modifies polymerase activity, such as in the fingers region. Preferably, the non-natural split in the polymerase results in split polymerases that have improved incorporation of a non-natural nucleotide or reduced discrimination, especially for dual-labeled nucleotide analogs. The split polymerase may be further modified to further improve incorporation of a non- natural nucleotide, to reduce 3 '-5' exonuclease activity, to reduce 5 '-3' exonuclease activity, to reduce uracil detection activity, to increase stability as compared to the non- split polymerase, or modified to incorporate a DNA binding domain. In preferred embodiments, the thermostable polymerases used to create the split polymerases are chosen from the Family A and Family B DNA polymerases. This includes, but is not limited to archaeal, mammalian, or bacteriophage polymerases. In one embodiment, the split polymerase is a thermostable archaeal polymerase which is comprised of one or more splits. In one embodiment, the split polymerase is a polymerase that has been split in the fingers domain.

[064] A split polymerase is understood to be a polymerase expressed as at least two discrete polypeptides (e.g., an N-terminal fragment and a C-terminal fragment) that associate to form a single, functional polymerase molecule. In general, the association is non-covalent, although covalent bridges, such as sulfur-sulfur bridges, may form to connect the polypeptides. While not required, expression of the fragments of a split polymerase is preferably translationally coupled to promote proper folding. A split polymerase includes at least one split, preferably in the fingers domain of the protein with one split being preferred. A split is understood as a break in the amino acid sequence of the polymerase such that at least two polypeptides compose the split polymerase. The Attorney Docket No: STG-188 Customer No.: 27,495 split can further result in the insertion, deletion, and/or mutation of amino acids at the site of the split. That is, if the polypeptides were joined, there would be a gap, insertion, and/or mutation as compared to the wild-type sequence at the site of the split. In preferred embodiments, up to about 5 amino acids can be deleted at the site of the split. Alternatively, in other preferred embodiments, up to about 40 amino acids can be added to either or both of the new C-terminus and/or the N-terminus created at the split. Frequently, if a methionine is not present in the wild-type coding sequence for the C- terminal fragment of the polymerase, one is added to the N-terminus of the coding sequence for the creation of a translational start site. The coding sequence for a methionine is inserted to promote ribosome reinitiation of translation in polycistronic mRNAs, or to allow initiation of translation of the C-terminal fragment from a second promoter, typically in a separate vector or expression construct. Alternatively, other initiation codons like GTG can also work in translational coupling. [065] Region III of various polymerases encompasses the "fingers domain," which consists of two long anti-parallel alpha helices, separated by a short loop (finger tip). An alignment of finger domains is shown in figures 1 and 7. The alpha helices comprising the fingers domain of archaeal DNA polymerases correspond to helices N/O in Pfu (Biles and Connolly, 2004, supra), R-RVS in Sso (Savino et al, 2004, Structure 12:2001) and OTP in Tgo DNA polymerase (Hopfner, Ct al., 1999 Proc. Natl. Acad. Sci USA, 96: 3600). The structures of several archaeal DNA polymerases have been determined, and the characteristics of the fingers domain have been discussed in several publications (Hopfner, et al 1999, supra; Zhao et al, 1999. Structure 7:1189; Rodriguez et al 2000, J. MoI. Biol. 299:447; Hashimoto et al, 2001, J. MoI. Biol. 306:469; Savino et al, 2004, supra). Comparison to the bacteriophage Rb69 DNA pol-primer/template-dNTP crystal structure, implicates the conserved amino acids in the fingers domain in dNTP binding and fidelity. However, the fingers domain of T4 and T4-like phages has an extended finger tip domain that is not found in the Archeal Family B polymerases. The fingers domain is highly conserved across archaea, suggesting that all DNA polymerases have a similar helix-loop-helix motif, and therefore use the same mechanisms for dNTP Attorney Docket No: STG-188 Customer No.: 27,495 recognition. Furthermore, the amino acid sequences of many polymerases are known, allowing for extrapolation of split sites and mutations from one polymerase to another. Folding of the helix-loop-helix leads to the proximal location of a number of highly conserved amino acids derived from both alpha helices.

[066] The fingers subdomain is formed primarily by two long antiparallel α helicies, which includes conserved regions III and IV. The length of the fingers domain varies substantially within the Family B polymerases. For example, some gp43 proteins from T4 and T4-like viruses RB69, RB49, RB43, and Aehl include a structurally disordered 50 to 75 amino acid residue sequence at the tip of the finger that diverges widely among the gp43 proteins (Petrov et al, 2006, J. MoI. Biol. 361:46). Also, interestingly, the Family B polymerases of some archaea resemble RB69 gp43 in global structure, but lack this finger tip feature entirely in their fingers domain. However, amino acids in the short intervening sequence between the helicies, especially amino acids 472 and 473 (Pfu numbering), have been shown to be involved in polymerase fidelity (Biles and Connolly, 2004. Nucleic Acids Res. 32:e 176). In other gp43 proteins, the coding sequence is interrupted at the fingertip sequence, thus splitting the gene into two separate ORFs (open reading frames) or cistrons, 43A and 43B. A natural split polymerase has also been identified in archaea; however, the split is located outside of the fingers domain (Kelman et al., 1999. J. Biol. Chem. 274:28751).

[067] As used herein, "Family A DNA polymerase" or "A Family DNA polymerase" or variation thereof refers to any DNA polymerase that is classified as a member of the Family A polymerases wherein the Family A classification is based on structural similarity to E. coli DNA polymerase I. Family A polymerases include, but are not limited to bacterial DNA polymerases such as E. coli DNA polymerase I, Streptococcus pneumoniae DNA polymerase I, Thermus aquaticus DNA polymerase I, Thermus flavus DNA polymerase I, Thermotoga maritima DNA polymerase I; bacteriophage DNA Polymerases T5 DNA polymerase, T7 DNA polymerase, Spol DNA polymerase, Spo2 DNA polymerase; yeast mitochondrial DNA polymerase II; and thermostable DNA polymerase from Thermus aquaticus (Taq). Attorney Docket No: STG-188 Customer No.: 27,495

[068] As used herein, "Family B DNA polymerase" or "B Family DNA polymerase" or variation thereof refers to any DNA polymerase that is classified as a member of the Family B DNA polymerases, where the Family B classification is based on structural similarity to E. coli DNA polymerase II. Family B DNA polymerases, formerly known as α-family polymerases (Braithwaite and Ito, 1991, supra), include, but are not limited to human α, δ and ε DNA polymerases, T4, RB69 and Φ29 bacteriophage DNA polymerases, and Pyrococcus furiosus DNA polymerase (Pfu polymerase) (see, e.g., Table I).

[069] Sequence comparison of the Family B DNA polymerases indicate six conserved regions I- VI, numbered in descending order of similarity (see, e.g., Braithwaite and Ito, 1993, supra, Edgell et al, 1997). Most of the conserved residues in the Family B polymerases are located within a radius of 10 A from the polymerase catalytic center as marked by the three most conserved carboxylates (Wang et al., 1997, Cell 89:1087). The three most highly conserved regions (designated I, II, and III) converge at the center of the active site from the palm (I), the fingers (II), and base of the thumb (III) to produce a contiguous conserved surface. An alignment of the fingers domain from a wide variety of Family B polymerases shows that this region is highly conserved (Figure 1). [070] Family B DNA polymerases can also be characterized by having a motif in the fingers domain having the sequence:

FIPSXLXXLXXXRQXXKXXMKXXXDPXEKXXLDYRQXAIKXLAN (SEQ ID NO: 5), wherein X is any amino acid. The sequence may be at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the motif. The cloned Family B DNA polymerases include, but are not limited to, Vent Thermococcus litoralis, Thermococcus sp. (Stral 1 ' 'T TY), Pab Pyrococcus abyssi, PYRHO Pyrococcus horikoshii, PYRSE Pyrococcus sp. (Strain GE23), Deep Vent Pyrococcus sp., Pfu Pyrococcus furiosus, JdF-3 Thermococcus sp., 9degN Thermococcus sp. (Strain 90N-7), KOD Pyrococcus sp., Tgo Thermococcus gorgonarius, THEFM Thermococcus fumicolans, METTH Methanobacterium thermoautotrophicum, Metj a Methanococcus jannaschii, POC Pyrodictium occultum, Apel Aeropyrum pernix, ARCFU Archaeoglobus fulgidus, Desulfurococcus sp. Tok, and Attorney Docket No: STG-188 Customer No.: 27,495

Thermococcus kodakarensis .

[071] An alignment of DNA polymerase protein sequences of each family, A, B, and C, across a broad spectrum of archaeal, bacterial, viral and eukaryotic organisms is presented in Braithwaite and Ito (1993), which is incorporated herein by reference. Recently, Family D (found in Euryarchaeota subdomain of Archaea), Family X (containing, for e.g., the well-known eukaryotic polymerase Pol β), and Family Y (comprised of translesion synthesis polymerases) have been described. Conserved motifs in DNA polymerase Family A, Family B, and reverse transcriptases include Motif A (Family A consensus sequence of DhSxIELR, Family B consensus of DhxSLYPS, reverse transcriptase consensus of Dh-GY) and Motif B (Family A consensus of GKxhNFGVLYG, Family B consensus of KhxxN-SLYG) (Gardner and Jack, 1999, Nucleic Acids Research, 27: 12, 2545-2553). The B-motif found in Family A and Family B DNA polymerases is also a conserved motif found in a broad range of DNA polymerases (Evans et al., 2000, Nucleic Acids Research 28:1059-1066). Amino acids important for primer-template interactions have also been identified in Family A and Family B polymerases (Hogg et al., 2004, EMBO, 23:1483-1493). Therefore, the split polymerases of the invention could be polymerases that are split in a conserved motif, other than the finger motif, of the Family A and Family B polymerases or any other polymerases that show the conserved motif.

[072] The split polymerases of the invention can increase incorporation of non-natural nucleotides and/or increase utilization of modified primer templates relative to the non- split polymerase. They are particularly suited for reduced discrimination of non-natural nucleotides, such as dual labeled nucleotide analogs. The non-natural nucleotide may be modified at the polyphosphate and/or at the base. The modification may comprise a fluorophore or a quencher. The labels include, but are not limited to, a fluorescent label, quencher, isotope, chemiluminescent label, quantum dot label, antigen, affinity moiety, or any other structure that allows detection of the nucleotide analog. In preferred embodiments, a quencher label is attached to the polyphosphate group and a fluorescent moiety is attached to the nucleobase or sugar of the nucleotide analog. Attorney Docket No: STG-188 Customer No.: 27,495

[073] The nucleobase moiety is preferably selected from the group consisting of adenine, cytosine, guanine, thymine, uracil and hypoxanthine, although modified forms and functional analogs of these are specifically contemplated. For example, non- conventional nucleotides, meaning a nucleotide structure that is not one of the four conventional deoxynucleotides dATP, dCTP, dGTP, and dTTP recognized by and incorporated by a DNA polymerase, are also envisioned to be applicable to the present invention. These include synthetic nucleotides, modified conventional nucleotides, ribonucleotides, and the like. Any one of the above non-conventional nucleotides may be a "conjugated nucleotide", which as used herein refers to nucleotides bearing a detectable label, including but not limited to a fluorescent label, quencher, isotope, chemiluminescent label, quantum dot label, antigen, or affinity moiety. Further, a dual- labeled nucleotide analog according to the invention may act as a chain terminator for the template-directed polymerization of a polynucleotide by a nucleic acid polymerase. [074] The split polymerase may be a chimeric polymerase, a combination of an N- terminal fragment from a first polymerase, and a C-terminal fragment of a second polymerase, preferably wherein the fragments are from the same family of polymerases (e.g. , both Family B polymerases). Any combination of polymerases may be used in the invention, as long as the combination includes at least two discrete polypeptides that associate to form a single, functional polymerase molecule.

[075] In the present invention, insertion of a split into the coding sequence may include the mutation of the coding sequence in at least one amino acid at the split site, typically to introduce an initiator methionine for reinitiation of translation of the C-terminal fragment of the split polymerase. Mutations may be introduced into coding sequences adjacent to the split site, especially downstream of the split site, to remove coding sequences for methionines, especially those in alternate reading frames. ATG methionine sequences in the two non-coding frames, which could compete for ribosome reinitiation, can often be removed by changing the wobble base of the codon in the correct reading frame. Methods for introducing mutations into a coding sequence without introducing mutations into the final amino acid sequence are well known to those skilled in the art. Attorney Docket No: STG-188 Customer No.: 27,495

[076] In another embodiment of the invention, the split results in the modification of coding sequence that results in the addition of coding sequence for at least one additional amino acid as compared to the wild-type sequence at the split site. The modification may result in the insertion of coding sequence for additional amino acids, or a modification to promote backsliding of the ribosome to a reinitiation codon upstream of the termination codon. The number of amino acids added in the final polymerase as compared to the wild type polymerase may be inserted is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, or 40 amino acids.

[077] In another embodiment of the invention, the split results in the deletion of coding sequence for at least one amino acid as compared to the wild-type sequence at the split site. The modification may result in the deletion of coding sequence for a number of amino acids, or a modification to promote sliding of the ribosome to a reinitiation codon downstream of the termination codon. The number of amino acids that may be deleted is at least 1, 2, 3, 4, or 5 amino acids.

[078] In an embodiment of the invention, the split polymerase further comprises one or more mutations, as compared to the wild-type coding sequence, in addition to any mutations caused by the split. Multiple mutations can be introduced to confer a single new property to the split polymerase, or multiple mutations can be introduced to confer multiple new properties on the polymerase. The mutations can be introduced in the finger region, close to or at where the split occurs, or can be added to regions farther away from the split.

[079] Mutations in the split polymerase can result in one or more changes in the enzyme. For example, mutations can increase the stability of the split polymerase as compared to a split polymerase not containing the mutation. In another example, the mutation can substantially reduce 3' to 5' exonuclease activity of the polymerase as compared to a polymerase not containing the mutation. In yet another example, the mutation further reduces the discrimination of the polymerase as compared to a polymerase not containing the mutation. This can result in reduced uracil detection and/or increased tendency to incorporate non-conventional nucleotides or nucleosides. In Attorney Docket No: STG-188 Customer No.: 27,495 another embodiment, the polymerase includes a non-sequence specific double stranded DNA binding domain attached to the polymerase, preferably at the C-terminus of the C- terminal fragment of the split polymerase, to increase the processivity of the polymerase as compared to a polymerase not having the domain.

[080] In an embodiment of the invention, the split polymerase is characterized by having a motif comprising RXXXK(X)nQXXXKXXXNSXGX (SEQ ID NO: 4), wherein X is any amino acid, and n= 15-80. In a further embodiment, the split lies within the motifof SEQ ID NO.: 4.

[081] In an embodiment of the invention, the split polymerase of the invention is characterized by having a motif comprising

FIPSXLXXLXXXRQXXKXXMKXXXDPXEKXXLDYRQXAIKXLAN (SEQ ID NO:5), wherein X is any amino acid. In a further embodiment, the split lies within the motif of SEQ ID NO.: 5. In an embodiment, the split lies within the region of amino acids 5 to 40 of SEQ ID NO: 5. In an embodiment, the split lies within the region of amino acids 5 to 30 of SEQ ID NO: 5. In an embodiment, the split lies within the region of amino acids 10 to 25 of SEQ ID NO: 5. In an embodiment, the split lies within the region of amino acids 15 to 25 of SEQ ID NO: 5.

[082] In an embodiment of the invention, the polymerase is characterized by having a motif comprising

FIPSBLXXLBXXRXXBKXZMKXJXDPBEKXBLDYRQZAIKBLAN (SEQ ID NO:6), wherein B is M, V, L, or I; J is S or T; U is F or Y; Z is Q, K, or R; and X is any amino acid. In a further embodiment, the split lies within the motif of SEQ ID NO.: 6. In an embodiment, the split lies within the region of amino acids 5 to 40 of SEQ ID NO:6. In an embodiment, the split lies within the region of amino acids 5 to 30 of SEQ ID NO: 6. In an embodiment, the split lies within the region of amino acids 10 to 25 of SEQ ID NO: 6. In an embodiment, the split lies within the region of amino acids 15 to 25 of SEQ ID NO: 6.

[083] In an embodiment of the invention, the split polymerase is characterized by having a motif comprising Attorney Docket No: STG-188 Customer No.: 27,495

GXXXXXLXXLXXXRXXXKXXMXXXXDXXXXXXLDXRQXAXKXXANXXYGY XXX

(SEQ ID NO: 53), wherein X is any amino acid, and n= 15-40 wherein X is any amino acid. In a further embodiment, the split lies within the motif of SEQ ID NO.: 53. In a further embodiment, the split lies within the motif of SEQ ID NO.: 53. In an embodiment, the split lies within the region of amino acids 5 to 40 of SEQ ID NO: 53. In an embodiment, the split lies within the region of amino acids 5 to 30 of SEQ ID NO: 53. In an embodiment, the split lies within the region of amino acids 10 to 25 of SEQ ID NO: 53. In an embodiment, the split lies within the region of amino acids 15 to 25 of SEQ ID NO: 53.

[084] In an embodiment of the invention, the split polymerase of the invention is characterized by having a motif comprising

GXXXXBLXXLBXXRXXBKXXMXXJXDXXOZXBLDXRQZABKBBANXUYGYX XX (SEQ ID NO: 54), wherein X is any amino acid. In a further embodiment, the split lies within the motif of SEQ ID NO.: 54. In an embodiment, the split lies within the region of amino acids 5 to 40 of SEQ ID NO: 54. In an embodiment, the split lies within the region of amino acids 5 to 30 of SEQ ID NO: 54. In an embodiment, the split lies within the region of amino acids 10 to 25 of SEQ ID NO: 54. In an embodiment, the split lies within the region of amino acids 15 to 25 of SEQ ID NO: 54. [085] In an embodiment of the invention, the spilt polymerase is characterized by having a motif comprising

GFIPSXLXXLXXXRQXXKXXMZXXXDPXXXXXLDYRQXAIKXLANSUYGYXX Y (SEQ ID NO: 55), wherein X is any amino acid, and n= 15-40 wherein X is any amino acid. In a further embodiment, the split lies within the motif of SEQ ID NO.: 55. In a further embodiment, the split lies within the motif of SEQ ID NO. : 55. In an embodiment, the split lies within the region of amino acids 5 to 40 of SEQ ID NO: 55. In an embodiment, the split lies within the region of amino acids 5 to 30 of SEQ ID NO: 55. In an embodiment, the split lies within the region of amino acids 10 to 25 of SEQ ID NO: 55. In an embodiment, the split lies within the region of amino acids 15 to 25 of SEQ ID Attorney Docket No: STG-188 Customer No.: 27,495

NO: 55.

[086] In an embodiment of the invention, the split polymerase of the invention is characterized by having a motif comprising

GFIPSBLXXLBXXRXXBKXXMZXJXDPBEZBBLD YRQZAIKBLANSUYGYXXY (SEQ ID NO: 56), wherein X is any amino acid. In a further embodiment, the split lies within the motif of SEQ ID NO.: 56. In an embodiment, the split lies within the region of amino acids 5 to 40 of SEQ ID NO: 56. In an embodiment, the split lies within the region of amino acids 5 to 30 of SEQ ID NO: 56. In an embodiment, the split lies within the region of amino acids 10 to 25 of SEQ ID NO: 56. In an embodiment, the split lies within the region of amino acids 15 to 25 of SEQ ID NO: 56. In an embodiment, there is an additional Q484R mutation in SEQ ID NO:56.

[087] In an embodiment of the invention, the polymerase is characterized by having a sequence that is at least 70% identical to SEQ ID NO: 4, 5, 6, 53, 54, 55, or 56, such as at least 75% identical, 80% identical, 85% identical, 90% identical, or 95% identical. In a further embodiment, the split lies within a sequence that is at least 70% identical to the motif of SEQ ID NO.: 4, 5, 6, 53, 54, 55, or 56, such as at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical. In an embodiment, the split lies within the region of amino acids 5 to 40 within a sequence that is at least 70% identical to the motif of SEQ ID NO: 5, 6, 53, 54, 55, or 56, such as at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical. In an embodiment, the split lies within the region of amino acids 5 to 30 within a sequence that is at least 70% identical to the motif of SEQ ID NO: 5 or 6, such as at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical. In an embodiment, the split lies within the region of amino acids 10 to 25 within a sequence that is at least 70% identical to the motif of SEQ ID NO: 5 or 6, such as at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical. In an embodiment, the split lies within the region of amino acids 15 to 25 within a sequence that is at least 70% identical to the motif of SEQ ID NO: 5, 6, 53, 54, 55, or 56, such as at least 75% Attorney Docket No: STG-188 Customer No.: 27,495 identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical. Of course the identity of any of these embodiments can fall within the range disclosed without having been particularly stated, such as 84% identical or 92% identical, etc. That is, the invention contemplates any particular value or range of values within the cited ranges, and each particular value need not be specifically recited herein for one of skill in the art to understand the collection of values and ranges so contemplated.

[088] In a further embodiment of the invention, the split is at a site within a region of Pfu DNA polymerase. Preferably, the split is introduced in the fingers domain of the polymerase. As examples, the split can be found to occur somewhere in the region of amino acids 448 to 500

(GFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGY), amino acids 460 to 480, amino acids 465 to 475, or amino acids 466 to 470 of Pfu DNA polymerase. In one embodiment, the split polymerase is located in the fingers domain and has at least 50% identity to amino acids 448 to 500 of the Pfu polymerase, such as at least 55% identity, at least 60% identity, at least 70% identity, at least 80% identity, or at least 90% identity.

[089] To summarize, in one embodiment of the invention, a purified thermostable polymerase is provided, wherein the polymerase is a split polymerase wherein the split is within the fingers domain within a region characterized by a motif comprising: FIPSBLXXLBXXRXXBKXZMKXJXDPBEKBLDYRQZAIKBLAN (SEQ ID NO: 6), wherein B is M, V, L, or I; J is S or T; U is F or Y; Z is Q, K, or R; and X is any amino acid. In an embodiment, the polymerase is selected from the group consisting of Thermococcus litoralis (Vent) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Thermococcus JdF-3 DNA polymerase, Pyrococcus horikoshii (Pho) DNA polymerase, Thermococcus gorgonarius (T go) DNA polymerase, Pyrococcus sp. GBD (Deep Vent) DNA polymerase, and archaeal polymerase I. The polymerase can be a DNA polymerase selected from the group consisting of Pyrococcus and Thermococcus. In an embodiment, the polymerase comprises a finger domain which has an amino acid Attorney Docket No: STG-188 Customer No.: 27,495 sequence at least 70%, at least 80%, or at least 90% identical to SEQ ID NO: 4, 5, or 6. [090] In an embodiment, the invention provides an isolated nucleic acid polymerase wherein the polymerase is a split Pyrococcus furiosus (Pfu) DNA polymerase comprising a fingers domain, and the split is within the fingers domain. The polymerase may be comprised of a split between amino acids 448 and 500 or amino acids 467 and 468. [091] In an embodiment, a split polymerase of the invention further comprises a mutation in the amino acid sequence as compared to the non-split polymerase. The mutation may be at an amino acid selected from the group consisting of L409, Y410, P411, R461, K465, Q472, Q484, A486, R488, L490, A 491, N492, Y495, and Y497 to reduce discrimination of the polymerase as compared to the polymerase without the mutation. The reduced discrimination may comprise reduced uracil detection or increased tendency to incorporate non-conventional nucleotides or nucleosides. The mutation is selected from the group consisting of L409H, Y41OV, P41 IL, R461A/N, K465A/N, Q472H, Q484R/K, A486T, R488A, L490Y, A491Y, N492A, Y495S, Y497A1L. The polymerase may further comprise a mutation at an amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting of D141A/E143A double mutation, and V93R, to reduce 3' to 5' exonuclease activity as compared to the polymerase without the mutation. The polymerase may further comprise a mutation at an amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting of A318, V604, and A662, to increase stability of the split polymerase as compared to the polymerase without the mutation. In another embodiment, the mutation increases the stability of the split polymerase to a level comparable to a non-split polymerase. The mutation may be selected from the group consisting of A318T, V604L, A662V, and any combination thereof. As an example, the polymerase may comprise a mutation at an amino acid site corresponding to amino acid Q484 in Pfu DNA polymerase. A preferred mutation according to the invention is Q484R. The polymerase may further comprise an amino acid site corresponding to a site in Pfu DNA polymerase selected from the group consisting of A318T, V604L, A662V, D141A, E143A, and V93R. Attorney Docket No: STG-188 Customer No.: 27,495

[092] In an embodiment, the polymerase further comprises a double stranded sequence- non-specific nucleic acid binding domain attached to the polymerase. The sequence-nonspecific nucleic acid binding domain may be an Sso7 derivative with less than 90% identity to wild type Sso7.

[093] In another embodiment of the invention, the invention provides a purified thermostable polymerase wherein the polymerase is a split polymerase, wherein the split is within a region characterized by a motif comprising:

GXXXXBLXXLBXXRXXBKXXMXXJXDXXOZXBLDXRQZABKBBANXUYGYX XX (SEQ ID NO: 54), wherein B is M, V, L, or I; J is S or T; O is D or E; U is F or Y; Z is Q, K, or R; and X is any amino acid. The polymerase may be selected from the group consisting of JdF-3 Thermococcus sp. DNA polymerase; Pyrococcus horikoshii (Pho) DNA polymerase; DNA polymerase 1 Pyrococcus abyssi (Pab polymerase); DNA- dependent DNA polymerase; endonuclease Pyrococcus sp.; Vent Thermococcus litoralis DNA polymerase; Deep Vent Pyrococcus sp DNA polymerase; 9°N Thermococcus sp. (9ON-7) DNA polymerase; Thermococcus gorgonarius (T go) DNA polymerase; Thermococcus aggregans (Tfu) DNA polymerase; Pyrococcus sp. ST700 (ST700) DNA polymerase; Pyrococcus furiosus (Pfu) DNA polymerase; DNA polymerase and endonucleases Thermococcus sp. GE8; Thermococcus litoralis DNA dependent DNA polymerase (TLI); DNA-dependent polymerase precursor Thermococcus sp. NAI (TSPNAl); DNA-directed DNA polymerase (EC 2.7.7.7) KOD, intein containing precursor - Pyrococcus sp. (strain KODl) (TSPKOD); PoIA precursor Thermococcus zilligii (TZI); Thermococcus kodakarensis (THY) DNA polymerase; Pyrococcus glycovorans (PGL) DNA polymerase; DNA polymerase II Sulfolobus tokodai str. 7 (SULFOTO) DNA polymerase; and Desulfurococcus sp. Tok (TOK) DNA polymerase. In one embodiment, the polymerase is an archaeal DNA polymerase selected from the group consisting of Pyrococcus and Thermococcus. The polymerase may comprise a finger domain which has an amino acid sequence at least 70% identical, at least 80% identical, or at least 90% identical to SEQ ID NO. 53, 54, 55, or 56. The polymerase may further comprise a mutation at an amino acid corresponding to a site in Pfu DNA Attorney Docket No: STG-188 Customer No.: 27,495 polymerase selected from the group consisting of L409, Y410, P411, R461, K465, Q472, Q484, A486, R488, L490, A491, N492, Y495, and Y497, to reduce discrimination of the polymerase as compared to the polymerase without the mutation. The polymerase may have reduced discrimination which may comprise reduced uracil detection or increased tendency to incorporate a non-conventional nucleotides or nucleosides. The polymerase may have a mutation selected from the group consisting of L409H, Y410V, P41 IL, R461AIN, K465A/N, Q472H, Q484R/K, A486T, R488A, L490Y, A491Y, N492A, Y495S, Y497A/L, and any combination thereof. The polymerase may have a mutation at an amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting of D141A/EI43A double mutation, and V93R further comprising at least one mutation to reduce 3' to 5' exonuclease activity as compared to the polymerase without the mutation. The polymerase may comprise a mutation at an amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting of A318, V604, and A662, to increase stability of the split polymerase as compared to the polymerase without the mutation. The mutation may increase the stability of the split polymerase to a level comparable to a non-split polymerase. The mutation may be selected from the group consisting of A318T, V604L, A662V, and any combination thereof. The polymerase may comprise a mutation at an amino acid site corresponding to amino acid Q484 in Pfu DNA polymerase, such as Q484R or Q484K. The polymerase may comprise a mutation at an amino acid site corresponding to a site in Pfu DNA polymerase selected from the group consisting of A318T, V604L, A662V, D141A, E143A, V93R, and a combination thereof. The polymerase may further comprise a double stranded sequence-non-specific nucleic acid binding domain attached to the polymerase. The sequence-non-specific nucleic acid binding domain may be an Sso7 derivative with less than 90% identity to wild type Sso7.

[094] In summary, an amino acid sequence of a split polymerase of the invention can be comprised of a mutation in at least one amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting of L409, Y410, P411, R461, K465, Q472, Q484, A486, R488, L490, A 491, N492, Y495, and Y497. An amino acid sequence of Attorney Docket No: STG-188 Customer No.: 27,495 the split polymerase may further be comprised of a mutation corresponding to a site in Pfu DNA polymerase that is selected from the group consisting of L409H, Y410V, P411L, R461A/N, K465A/N, Q472H, Q484R/K, A486T, R488A, L490Y, A491Y, N492A, Y495S, Y497A/L, and any combination thereof. In another embodiment, the amino acid sequence of a split polymerase of the invention is comprised of the double mutation D141A/E143A, single mutation V93R, or both, corresponding to a site in Pfu DNA polymerase, to reduce 3' to 5' exonuclease activity as compared to the polymerase without the mutation. In an embodiment, the amino acid sequence of the polymerase further comprises a mutation at an amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting of A318, V604, A662, and any combination thereof, to increase stability of the split polymerase as compared to the polymerase without the mutation. The amino acid sequence of the split polymerase may further be comprised of a C-terminal fusion with a DNA binding protein. [095] In one exemplary example, the split polymerase is the SQ split polymerase. This mutant is comprised of a seamless split after amino acid 467 to create a fragment encompassing amino acids 1-467 and a fragment encompassing amino acids 468-775 with the additional mutation Q484R. SQ may be further modified to eliminate exonuclease activity by adding D141 A/E143A mutations (Fig. 13). SQ is not comprised of the additional mutations found in mutant 4Cl 1 (1-467 V93R/A318T)/(468-775 V604L/A662V-Ss07d7m). The nucleic acid sequence of the exo- mutant SQ is also disclosed (SEQ ID NO:93). The exo- SQ mutant was found to work as well in non- conventional nucleotide incorporation assays as the 4Cl 1 mutant. Because the exo- SQ mutant is only comprised of the seamless split, Q484R, and the exo-minus (D141A/E143A) mutations, in one embodiment, the present invention provides this set of mutations as a way of increasing non-conventional nucleotide incorporation. Herein, "exo-" or "exo-minus" refers to a mutant that does not have exonuclease activity. [096] In another general aspect, the present invention provides nucleic acids encoding split polymerases of the invention. Because split polymerases are only a part or portion of a complete polymerase sequence, the nucleic acids of the invention only encode for a Attorney Docket No: STG-188 Customer No.: 27,495 part or portion of a polymerase in any one reading frame. Split polymerases are preferably expressed from a polycistronic, preferably bicistronic, operon in which the expression of the N-terminal polypeptide portion (fragment I) and the C-terminal polypeptide portion (fragment II) of the polymerase are translationally coupled. In another embodiment, the split polymerase is encoded by two or more coding sequences that are present on two or more separate plasmids in the same host cell, or in two separate host cells.

[097] A coding sequence or mRNA is said to be polycistronic when it contains the genetic information to translate more than one polypeptide from a single promoter typically without disengagement of the ribosome from the mRNA. A polycistronic mRNA is composed of at least two cistrons. As used herein, a cistron is a segment of DNA that specifies a single polypeptide unit. The polypeptides expressed from a polycistronic mRNA may or may not form a complex after translation. Most of the mRNA found in prokaryotes is polycistronic.

[098] Translationally coupled is understood to mean that translation of multiple portions of the split polymerase are translated in a coordinated manner that may allow association of the subunits at the time of translation. In other words, "translationally coupled" is understood to mean that the ribosome encounters a stop codon, terminates translation but does not disengage from the RNA, and scans for, finds, and initiates translation again at a nearby ATG. Coding sequences from each of the subunits may be under the control of the same inducible promoter to allow for coordinated expression. Alternatively, the fragments may be coded for by a single, polycistronic mRNA.

[099] In one embodiment of the invention the split is a seamless split wherein no new amino acids are introduced or deleted at the split site as compared to the wild-type coding sequence. For example, the split in the 4Cl 1 mutant (shown in Figure 3B) deleted an "A" from the reading frame (AAAATGA). Fortuitously, the "A" from the downstream ATG maintained the previous codon (AAA) allowing conservation of the polymerase amino acid sequence. The second codon in the frameshift was recognized as TGA which terminates translation. Translational reinitiation at the ATG, one base upstream from the Attorney Docket No: STG-188 Customer No.: 27,495

TGA, corrected the frameshift, producing a second protein fragment, which in complementation with the first fragment preserved the amino acid of the polymerase. In this way, through careful design using methods well known to those skilled in the art, a seamless split can be introduced so that there is no alteration of the polypeptide sequence. However, the invention provides for nucleic acids that are different from the wild-type nucleic acid sequence. For example, nucleotide substitutions can be made that change the nucleic acid sequence, but do not change the polypeptide sequence. [100] Other designs are also envisioned in the present invention, such as splits that are not seamless. In this embodiment, a frameshift creates a termination codon (TAA, TAG or TGA) in addition to shifting the protein-coding region out of frame, such that additional amino acids are introduced. The termination codon terminates translation of the first fragment, but translation can begin again if the ribosome can find a reinitiation codon within seven (upstream or downstream) codons or as far away as 46 nucleotides upstream or downstream (Karamyshev, 2004, supra). Positioning initiating codons further away is possible, but less preferred due to a likely decrease in efficiency of translation of fragment II. The reinitiating codon is usually an ATG, but can be ACG (Thr), CTG (Leu) and GTG (VaI). Even if the initiation codon encodes another amino acid, methionine is inserted into the first position of the peptide chain. Of course, in these designs, the invention provides for nucleic acid sequences that are comprised of additions or deletions as compared to the wild-type nucleic acid sequence. While not critical to practice of the invention, it is often desirable to express the fragments making up the split polymerase in a manner that not only provides similar molar amounts of each fragment, but also results in production of each fragment in a temporally proximate manner. While not being limited to any one mechanism of action, it is thought that these considerations allow for proper production and folding of the various fragments to create a functional end product.

[101] Some designs may require one or more amino acid replacements to introduce an initiating methionine. It is preferred that out of frame ATG triplets other than the one for the desired reinitiating methionine be altered to prevent incorrect reinitiation of Attorney Docket No: STG-188 Customer No.: 27,495 translation. Methods of altering nucleotide sequence without altering amino acid sequence are well known to those skilled in the art. It may also be beneficial to mutate non-initiator methionines in the coding frame to introduce conservative mutations into the amino acid sequence. The relative advantages and disadvantages of altering methionines encoded in frame are well within the ability of those skilled in the art. [102] Splits that are not seamless will result in split polymerases that contain mutations. Therefore, the present invention provides mutant split polymerases that comprise mutations in one or more amino acids. These mutations may be added to the coding sequence to generate functional segments of the split polymerase. Mutations may also be added to allow better and/or different activity of the split polymerase and may be found in a region different from the split site. Mutations may be added to the wild type sequence of a polymerase to increase the stability of the split polymerase, to reduce 3' to 5' exonuclease activity, to reduce discrimination of the polymerase, and/or any other characteristic that may be beneficial for the use of the split polymerase. The mutations can be chosen depending on which characteristics are desired for the split polymerase and any combination of mutations can be found in one split polymerase. [103] In a preferred embodiment, the split polymerases of the invention are encoded by a polycistronic operon in which expression of each portion or cistron of the split polymerase is translationally coupled to translation of at least one other portion of the split polymerase. In one embodiment, the polycistronic coding sequence contains at least one ribosomal reinitiation signal proximal to a frameshift termination codon. [104] In an alternative embodiment, the split polymerases of the invention are encoded by two coding sequences that are present on two separate plasmids in the same host cell, or in two separate host cells. If the two plasmids are contained within a single host cell, the same inducible promoter (e.g., tetracycline or IPTG inducible) can be used to promote coordinated expression of the fragments of the split polymerase. However, it may be beneficial to use two different inducible promoters. For example, there may be cases where one fragment of the split polymerase is less stable than another and, therefore, requires a stronger promoter so that more of the less stable fragment is produced. It may Attorney Docket No: STG-188 Customer No.: 27,495 also be beneficial in some cases to use a constitutive promoter, depending on the conditions for expression of the split polymerase.

[105] In summary, the present invention provides a nucleic acid encoding for a part or all of a split polymerase. In one embodiment, the split may be in the fingers domain. In one embodiment, the nucleic acid may be further comprised of at least one mutation that improves incorporation of a non-natural nucleotide, reduces 3 '-5' exonuclease activity, reduces 5 '-3' exonuclease activity, reduces uracil detection activity, or increases stability when expressed as a split polymerase as compared to the non-split polymerase. In another embodiment, the nucleic acid is comprised of at least one mutation such that the split polymerase expressed from the nucleic acid comprises a mutation in at least one amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting of L409, Y410, P411, R461, K465, Q472, Q484, A486, R488, L490, A 491, N492, Y495, Y497, D141, E143, V93, A318, V604, and A662.

[106] In a further general aspect, the present invention provides methods of making a split polymerase of the invention. In general, the methods comprise providing at least two polymerase coding regions which are operably linked to at least one promoter, introducing the coding regions into a system in which the regions can be expressed, such as in at least one host cell or in an in vitro expression system, and incubating the system under conditions allowing the formation of a functional split polymerase. Stated another way, in embodiments, the methods comprise introducing at least two coding sequences encoding part of a polymerase into at least one host cell, and incubating the host cell under conditions allowing the translation of the coding regions and assembly of the translated products into a functional split polymerase in vivo. Optionally, the assembled split polymerase can be purified and/or tested for activity. Alternatively, the method may comprise assembly of the translated products into a functional split polymerase after purification of the split polymerase polypeptides from the host cells. In this case, assembly would take place outside of the host cell or in vitro. [107] In a preferred embodiment, the method comprises providing a polycistronic coding region sequence encoding a split polymerase, introducing the coding regions into Attorney Docket No: STG-188 Customer No.: 27,495 a host cell, and culturing the cell under conditions which permit production of the split polymerase. Optionally, the split polymerase may be purified before or after assembly. Also optionally, the split polymerase produced by the methods of the invention can be tested for polymerase activity and/or can be tested for non-conventional nucleotide incorporation. In view of this aspect of the invention, it should be apparent that the invention encompasses recombinant cells comprising nucleotides encoding split polymerases, and recombinant cells comprising non-naturally occurring split polymerases.

[108] The methods may comprise introducing into a host cell a nucleic acid comprising a polycistronic coding region sequence encoding a polymerase wherein the coding region is operably linked to a promoter, and culturing the cell under conditions that permit production of the polymerase. The host cell may be an E. coli or of the genus Thermococcus or Pyrococcus. In one embodiment, the method is a method of decreasing discrimination of a polymerase by introducing a split into a region of the polymerase characterized by a motif comprising:

FIPSBLXXLBXXRXXBKXZMKXJXDPBEKXBLDYRQZAIKBLAN (SEQ ID NO: 6), wherein B is M, V, L, or I; J is S or T; U is F or Y; Z is Q, K, or R; and X is any amino acid. The polymerase may be selected from the group consisting of Thermococcus litoralis (Vent) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Thermococcus JdF-3 DNA polymerase, Pyrococcus horikoshii (Pho) DNA polymerase, Thermococcus gorgonarius (T go) DNA polymerase, Pyrococcus sp. GBD (Deep Vent) DNA polymerase, and archaeal polymerase I. In one embodiment, the DNA polymerase is selected from the group consisting of Pyrococcus and Thermococcus, such as Thermococcus litoralis (Vent) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, Thermococcus JdF-3 DNA polymerase, Pyrococcus horikoshii (Pho) DNA polymerase, Thermococcus gorgonarius (T go) DNA polymerase, Pyrococcus sp. GBD (Deep Vent) DNA polymerase, and archaeal polymerase I. In one embodiment, the polymerase is a split Pyrococcus furiosus (Pfu) DNA polymerase. The split may be between amino acids 448 and 500, or 467 and 468. Attorney Docket No: STG-188 Customer No.: 27,495

[109] The methods may include a polymerase that has a mutation at an amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting of L409, Y41O, P411, R461, K465, Q472, Q484, A486, R488, L490, A 491, N492, Y495, and Y497 to reduce discrimination of the polymerase as compared to the polymerase without the mutation. The reduced discrimination may comprise reduced uracil detection or increased tendency to incorporate a non-conventional nucleotide. The mutation may be selected from the group consisting of L409H, Y4I0V, P41 IL, R46IA/N, K465A/N, Q472H, Q484R/K, A486T, R488A, L490Y, A491 Y, N492A, Y495S, and Y497A/L. In another embodiment, the polymerase may further comprise a mutation at an amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting of D141A/E143A double mutation, and V93R, to reduce 3' to 5' exonuclease activity as compared to the polymerase without the mutation. In another embodiment, the polymerase may comprise a mutation at an amino acid corresponding to a site in Pfu DNA polymerase selected from the group consisting ofA318, V604 and A662 to increase stability of the split polymerase as compared to the polymerase without the mutation. In one embodiment, the mutation increases the stability of the split polymerase to a level comparable to a non-split polymerase. Also, the mutation may be selected from the group consisting of A318T, V604L, and A662V. In preferred embodiment, the polymerase further comprises a mutation at an amino acid site corresponding to amino acid Q484 in Phi DNA polymerase, such as Q484R.

The methods may further comprise a polymerase with at least one mutation at an amino acid site corresponding to a site in Pfu DNA polymerase selected from the group consisting of A318T, V604L, A662V, D141A, E143A, and V93R. In one embodiment, the methods comprise a polymerase with a double stranded sequence-non-specific nucleic acid binding domain attached to the polymerase. The sequence-non-specific nucleic acid binding domain may be an Sso7 derivative with less than 90% identity to wild type Sso7. The methods of making a split polymerase of the invention are envisioned to comprise no additional mutations (other than any formed from making the split), one mutation, or more than one mutation. The split polymerase may be one wherein the polymerase is Attorney Docket No: STG-188 Customer No.: 27,495 further modified to further improve incorporation of a non-natural nucleotide, to reduce 3 '-5' exonuclease activity, to reduce 5 '-3' exonuclease activity, to reduce uracil detection activity, to increase stability as compared to the non-split polymerase, or modified to incorporate a DNA binding domain.

[110] In a further aspect, the invention provides methods of using the split polymerase of the invention. A split polymerase of the invention can be used for nucleic acid amplification, nucleic acid sequencing, quantitative PCR (QPCR), nucleic acid labeling, for synthesis with modified primers or templates, or in any reaction that requires a polymerase. The invention is particularly suited for methods that involve non- conventional nucleotides and nucleotide analogs, such as dual-labeled nucleotide analogs. In another embodiment, the invention can be used to develop complementation systems where a polymerase signal is generated when two polymerase fragments that are expressed separately assemble together. In this case, one polymerase fragment could be used as a bait to screen a library fused to the other polymerase fragment. [Ill] The methods may comprise: (a) providing a DNA polymerase of the invention and (b) contacting the polymerase with a nucleic acid template, wherein the polymerase permits DNA synthesis. In another embodiment, the methods include a method for DNA synthesis with reduced discrimination as compared to synthesis with a wild type DNA polymerase comprising: (a) providing a DNA polymerase of the invention and (b) contacting the polymerase with a nucleic acid template and non-conventional nucleotides, wherein the polymerase permits DNA synthesis. The method may be a method for cloning of a DNA synthesis product comprising: (a) providing DNA polymerase of the invention and (b) contacting the DNA polymerase with a nucleic acid template, wherein the archaeal DNA polymerase permits DNA synthesis to generate a synthesized DNA product and (c) inserting said synthesized DNA product into a cloning vector. In another embodiment, the methods comprise: (a) providing a DNA polymerase of the invention; (b) generating chain terminated fragments from the DNA template to be sequenced with the DNA polymerase in the presence of at least one chain terminating agent and one or more nucleotide triphosphates, and (c) determining the sequence of the DNA from the Attorney Docket No: STG-188 Customer No.: 27,495 sizes of the fragments. The method may be a method of DNA synthesis with increased incorporation of dual-labeled nucleotides as compared to synthesis with a wild type DNA polymerase comprising: (a) providing a DNA polymerase of the invention and (b) contacting the polymerase with a nucleic acid template and dual-labeled nucleotides, wherein the polymerase permits DNA synthesis. The method may be a method of quantitative PCR (QPCR) with increased incorporation of dual-labeled nucleotides as compared to QPCR with a wild type DNA polymerase comprising (a) providing a DNA polymerase of the invention; and (b) contacting the polymerase with a nucleic acid template and dual-labeled nucleotides, wherein the polymerase permits DNA synthesis. In some embodiments, the methods are performed in the absence of a reducing agent, such as dithiothreitol (DTT) or β-mercaptoethanol, to improve stability of the split polymerase.

[112] In another embodiment, the methods of the invention are used to increase uptake of non-natural nucleotides. This can be accomplished by splitting a polymerase in such a way that the polymerase shows increased activity for non-natural nucleotides. Higher nucleotide analog uptake can also be increased by combining the split with other mutations in the amino acid sequence compared to using the split or the amino acid replacement alone.

[113] The invention provides for a method for DNA synthesis comprising providing a non-natural split DNA polymerase, and contacting the polymerase with a nucleic acid template, wherein the polymerase permits DNA synthesis. In one embodiment, the method of DNA synthesis is a method that comprises a split polymerase with increased ability to incorporate non-natural nucleotides or increased ability to utilize modified primer templates relative to the non-split polymerase. The method may be a method of DNA synthesis such as DNA sequencing, quantitative PCR (QPCR), DNA labeling, or a combination thereof. Practice of methods of the invention gives rise to numerous compositions comprising proteins and/or nucleic acids of the invention. [114] In an additional general aspect, the present invention provides compositions and kits comprising at least one split polymerase of the invention. Compositions comprise at Attorney Docket No: STG-188 Customer No.: 27,495 least one molecule of split polymerase or a nucleic acid of the invention. The amount of split polymerase or nucleic acid in the composition can vary widely, but generally the amount is sufficient to perform at least one method or assay of the invention, such as one that uses the split polymerase. The method or assay may involve nucleic acid sequencing, amplification, QPCR, or any other method that involves the use of a nucleic acid polymerase. In a preferred embodiment, the composition excludes reducing agents, such as DTT or β-mercaptoethanol, to better stabilize the split polymerase enzyme of the invention.

[115] In general, a kit according to the invention contains some or all of the components, reagents, supplies, etc. to practice a method according to the invention. In kits comprising a split polymerase according to the invention, the kit typically comprises a sufficient amount of split polymerase to allow at least one reaction involving the split polymerase to occur under appropriate conditions. In one embodiment, a kit contains at least one container (e.g., vial, tube, ampoule) containing a non-natural split polymerase. In embodiments, the kit further comprises at least one other substance used in a nucleic acid polymerase reaction (e.g., amplification, sequencing). Typically, the split polymerase will be supplied in one or more container, each container containing a sufficient amount of split polymerase to allow at least one reaction involving the split polymerase to occur. Kits may also comprise one or more other substances, which are typically substances that aid in the methods of the invention. These may include sterilized water, buffers, dNTPs, control DNA, primers, etc.

EXAMPLES [116] The invention will be further explained by the following Examples, which are intended to be purely exemplary of the invention, and should not be considered as limiting the invention in any way.

[117] Example 1 : Preparation of Split Pfu Polymerases by Site-Directed Mutagenesis and Affinity-Tag Purification

[118] Splits were introduced into Pfu DNA polymerase by site specific mutagenesis Attorney Docket No: STG-188 Customer No.: 27,495 using the QuikChange® Multi Site-Directed Mutagenesis Kit from Stratagene according to the Manufacturer's instructions. The primers used are shown in Table I.

Table I

[119] To make clones 105, 106 and 107, the primers above were used with a plasmid template encoding Pfu D141A/E143A (exo-) DNA polymerase with a C-terminal 6X histidine affinity tag. The 108, 109 and 110 clones were made using a similar DNA template that further contained a Q484R mutation in the Pfu DNA polymerase sequence. The split site in clone 105 was moved one residue up, so that fragment I of clone 105 corresponds to Pfu DNA polymerase residues 1-466 as compared to fragment I of 4Cl 1 which encompasses residues 1-467. In clone 105, what was formerly lysine 467 of fragment I (in 4C 11) is now found as the second residue of fragment II. Since the new split site could not be engineered without adding a methionine for reinitiation, an additional methionine is also present in clone 105. So by reference to the wild-type Pfu amino acid sequence, the resulting Split Pfu 105 mutant can be notated as: I (1-466)/ II (M-467-775). Clone 106 was designed to use the same split and added methionine as clone 105, but additionally has a M3K mutation in fragment II. Therefore, the Split Pfu 106 mutant can be described as: I (1-466)/ II (K467M-M468K-(469-775) by reference to wild type Pfu. Fragment I of clone 107 is terminated at the equivalent of Pfu Tyr 546. Sequence was reinitiated with a non-native methionine and enhanced with an upstream ribosomal binding domain. Thus, Split Pfu 107 can be notated as: I (l-546)/II (M-547- 775). All three splits were planned individually and in combination with the Q484R mutation that further enhances FCD uptake in 4Cl 1 (numbering relative to Pfu DNA polymerase sequence). Those clones were named: 108 (105+Q484R), 109 (106 +Q484R) Attorney Docket No: STG-188 Customer No.: 27,495 and 110 (107+Q484R). Figure 3 depicts the relevant amino acid sequence of wild type Pfu DNA polymerase (Panel A), clone 4Cl 1 (Panel B), clones 105 and 108 (Panel C), clones 106 and 109 (Panel D) and clones 107 and 110 (Panel E).

[120] The reaction was composed of:

2.5 μl of 10x QuikChange Multi reaction buffer

18.5 μl H2O

1 μl plasmid template

1 μl dNTP mix

1 μl QuikChange Multi enzyme blend

1 μl plasmid template

[121] Cycling parameters were:

1 cycle 95°C 1 min 30 cycles 95°C 1 min 500C 1 min 65°C 17 min

[122] The reaction was treated with the restriction enzyme Dpn I at 37°C for one hour to remove the parental plasmid. Two microliters of the reaction was transformed into XLlO Gold competent cells and plated on LBamp50 plates for 20 hours at 300C. Colonies were picked and used to start 5ml overnight cultures in LBamp50 media at 300C overnight. Plasmid DNA was isolated from the cells using the Strataprep® Plasmid Miniprep Kit. The prepared plasmid was sequenced to confirm the incorporation of the desired mutations. Positives were transformed into BL21-codonPlus®(DE3)-RIPL Competent Cells (Stratagene) cells and plated on LBamp50 plates overnight at 300C for 16 hours. Colonies were picked and grown overnight at 300C in LBamp50 media. After 16 hours, one ml of media was used to start a culture in 50ml of LBamp50. The cells were grown Attorney Docket No: STG-188 Customer No.: 27,495 at 300C until the OD=6oo reached a value of 0.8 to 1.0, and then induced with a final concentration of ImM IPTG. The induced cells were shaken at 300C for 2-4 hours before being collected by centrifugation and frozen at -200C.

[123] Purification of His-tagged split Pfu polymerases was performed using Ni-NTA slurry from Qiagen and following the instructions for E. coli cells in the QiaExpressionist Manual protocols 9 and 12 (Qiagen 6/2003). Seventy- five μl of the eluted DNA polymerase was run through a Micro Bio-Spin 6 (BioRad) desalting column and into a buffer consisting of (final) 5OmM Tris-Cl pH 8.2, 0.1 mM EDTA, 10OmM KCl.

[124] Example 2: Purification of Split Pfu

[125] Exo-minus Pfu 4Cl 1 (1-467 V93R/D141A/E143A /A318T)/(468-775 Q484R/V604L/A662V-Ss07d7m)/ DNA polymerase was expressed in E. coli from a pET 11 vector. Induced cells were pelleted and resuspended in 40 mM Tris-Cl, ImM EDTA, pH 7.5. Protease inhibitors, lysozyme (-0.25 mg/ml), and 2-mercaptoethanol (10 mM) were added. Cells were lysed by sonication. The extract was heat treated (85°C, 15 min.) to denature heat-labile proteins. NaCl was added to IM, and polyethyleneimine to 0.175% to precipitate primarily contaminating nucleic acids. Precipitated material was removed by centrifugation. The supernatant was brought to 65% saturation with ammonium sulfate. Precipitated proteins were collected by centrifugation, washed once with 65% saturated ammonium sulfate, then dissolved in Buffer A-I (40 mM Tris-Cl, 1 mM EDTA, 10 mM 2-mercaptoethanol, pH 7.5). This was dialyzed overnight against Buffer A-I and then loaded on Q-Sepharose FF that had been equilibrated with Buffer A- 1. Flow-thru fractions containing exo- 4Cl 1 were collected and loaded directly on a SP- Sepharose HP column also equilibrated with Buffer A-I . After washing with BufferA-1 , exo- Pfu 4Cl 1 was eluted as the major peak with a 30 column- volume gradient to 500 mM KCl. The peak was pooled and dialyzed overnight back into Buffer A-I . This was loaded on a Heparin Sepharose HP column equilibrated in Buffer A-I . After washing with Buffer A-I, exo-4Cl 1 was again eluted as the major peak with a 20 column-volume gradient to 750 mM KCl. The peak was pooled, concentrated, and dialyzed into final Attorney Docket No: STG-188 Customer No.: 27,495 dialysis buffer (5OmM Tris-Cl, 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 50% glycerol, pH 8.2), and stored at -200C.

[126] Example 3 : Cloning, Expression, and Purification of Separate Pfu Fragments Vector Preparation

[127] The pET21b vector was prepared by amplifying the supercoiled plasmid vector with inverse primers that produced a linear molecule similar to one cut with EcoRI.

[128] Nine 50μl reactions were set up using the following parameters:

5 μl 10x PfuUltra™ II Fusion HS DNA Polymerase (Stratagene)

0.5 μl 100 mM dNTPs (25mM each, Stratagene) 1.25 μl forward primer ( 1 OOμM) 1.25 μl reverse primer ( 1 OOμM) 0.2 μl plasmid DNA (~0.2ng)

1 μl PfuUltra1"1 1

40. ,8 μl H2O

[129] Cycling parameters were:

J_c ycle

95( 3C 1 min

30 cycles

95' 3C 45 sec

50( 3C 45 sec

72( 3C 2 min

J_c ycle

72' 3C 5 min

[130] After amplification, the nine replicates were combined and treated with 5 μl of Dpn I restriction enzyme (Stratagene 4u/μl) for one hour at 37°C to destroy the plasmid Attorney Docket No: STG-188 Customer No.: 27,495 template. The DNA was purified with StrataPrep PCR kit (Stratagene) according to the manufacturer's recommended protocol.

[131] 1 μg of the purified linear vector DNA (in a total volume of 50μl) was combined with 5.5 μl Xi-Clone Buffer and 2 μl of Xi-Clone enzyme (Genlantis) for 30 minutes at room temperature. The reaction was purified and concentrated with a DNA Cleanup Spin Column (Genlantis) and stored at 200C.

Insert Preparation

[132] The two Pfu DNA polymerase fragments, fragment I (Pfu 1-467, D141A,E143A, A318T) and fragment II (Pfu 468-775 Q484R, V604L/A662V), were expressed separately by cloning into pET21b. Forward and reverse primers used are shown in Table II below.

Table II

Pfu Frag I For TTGTTTAACTTTAAGAAGGAGATATACATATGATT TTA GAT GTG GAT TAC ATA ACT GAA GAA GGA AAA C

Pfu Frag I Rev CCGGATCTCAGTGGTGGTGGTGGTGGTGTTT TGT CTT AAT CTT TTG TCT TTC CTC TAA CAA ATG TCC C

Pfu Frag 2 For TTGTTTAACTTTAAGAAGGAGATATACATATGAAG GAA ACT CAA GAT CCT ATA GAA AAA ATA CTC C

Pfu Frag 2 Rev CCGGATCTCAGTGGTGGTGGTGGTGGTG TCT CTT TTG CTT TTC TAA CAT GTC TAC TAG TTC TTT TGG

[133] The products were amplified from the 4C 11 plasmid with the following primers and method.

5 μl 1Ox PfuUltra™ II Fusion HS DNA Polymerase (Stratagene)

0.5 μl 100 mM dNTPs (25mM each, Stratagene)

2 μl forward primer (50ng/μl)

2 μl reverse primer (50ng/μl)

1 μl plasmid DNA (~0.2ng)

1 μl PfuUltra™ II Fusion HS DNA Polymerase (Stratagene)

38.5 μl H2O Attorney Docket No: STG-188 Customer No.: 27,495

[134] Two 50 μl reactions were amplified for each fragment as follows: 1 cycle 95°C l min 30 cycles 95°C 45 sec 500C 45 sec 72°C 30 sec 1 cycle 72°C 5 min

[135] After amplification, 4 units of the restriction enzyme Dpn I (Stratagene, 4u/μl) was added to each PCR amplification reaction to digest the plasmid template. The DNA was purified with StrataPrep PCR (Stratagene).

[136] 2μl of the prepared pET21 b vector was added to 10 μl of purified insert and then transformed into 50 μl of XLIO-Gold® Ultracompetent Cells (Stratagene). The transformed cells were plated on LB ampicillin (50 μg/ml) and incubated at 300C for 18 hours. Colonies were screened for the presence of insert by PCR amplification, and the insertions were verified by DNA sequencing.

[137] Purification of the Pfu fragments was attempted using the procedure described in Example 2. When purified using the same method, fragment I exhibited a different elution profile compared to intact Pfu DNA polymerase, consistent with the differences in isoelectric point. Fragment II was extremely insoluble and was not loaded onto any columns.

[138] Example 4: Assaying Nucleotide Analog incorporation by Endpoint PCR [139] To test the ability of split Pfu enzymes to perform in PCR with and without nucleotide analogs, a simple plasmid template was used (plasmid 2-20). This plasmid contained a ~800bp insert which was amplified out of the pET vector by primers on Attorney Docket No: STG-188 Customer No.: 27,495 either side of the vector insertion site.

[140] PCR reaction mixtures designed to test FCD utilization consisted of the following:

1.2 μl 1Ox cloned Pfu buffer (Stratagene)

0.24 μl 1OmM dATP, dGTP, TTP

0.29 μl Petrev primer (1 OμM)

0.29 μl Petfor primer (1 OμM)

0.5 μl diluted plasmid 2-20

1.0 μl of polymerase

1.0 μl of 600μM (total) dCTP/FCD mix (50% dCTP, 50% FCD)

7.48 μl H20

[141] The enzyme was tested in the above PCR at several dilutions to find the optimal volume to use in a PCR reaction. The optimal value determined was used in comparison with wild-type exo-Pfu and other mutants.

[142] The PCR reactions were amplified with the following parameters: 1 cycle 95°C 1 min 30 cycles 95°C 45 sec 58°C 45 sec 72°C 1 min 1 cycle 72°C 5 min

[143] When FCD is incorporated by a DNA polymerase, FAM-dCMP is added to the growing chain concomitant with the release of Dabcyl-pyrophosphate (PP). In solution or homogeneous assays (e.g., QPCR), fluorescence increases in direct proportion to the number of FAM-dCMP molecules incorporated, or the number of unquenched FAM moieties. Incorporation of analog was demonstrated for some of the split polymerases by Attorney Docket No: STG-188 Customer No.: 27,495 this method.

[144] Incorporation of analog was assessed by performing endpoint PCR, followed by gel electrophoresis and detection of fluorescent amplicon. The entire PCR reaction mixtures were run on a 1% agarose, IxTBE gel (Wide Mini ReadyAgarose Gel, 1 X TBE, 1%, 20 well, 15 x 10 cm, Bio-Rad Laboratories) and the FAM label was detected with ultraviolet light and a green filter. A visible band at the appropriate size results in the scoring of "yes" in Table III under analog incorporation. After recording the image, the gel was stained with ethidium bromide to visualize all DNA products. [145] To determine if the split polymerase was functional for incorporation of conventional nucleotides, a PCR reaction was run essentially under the conditions above with equal amounts of all four dNTPs and no nucleotide analogs. Detection limit of the gel was about 1 ng of nucleic acid per band. A visible band at the appropriate size results in the scoring of "yes" in Table VI under conventional nucleotide incorporation.

[146] Example 5: Preparation of Split Chimeric JdF-3/Pfu Polymerases

[147] Chimera polymerases were constructed from JdF-3 DNA polymerase and Pfu

DNA polymerase. The split was homologous to the split of 4Cl 1 in Pfu DNA polymerase.

Fragment I was encoded from the JdF-3 DNA polymerase gene while fragment II was encoded by Pfu DNA polymerase gene. The construct was engineered by making two

PCR products corresponding to the first fragment and the second fragment, purifying the products and then fusing the products by splice overlap. Fragment I ended at the same amino acid as the split JdF-3 Fragment I shown in Figure 4 (Panel B) and Fragment II started at M468 of Pfu DNA polymerase (seen in Figure 3).

[148] In addition to JdF-3 sequence specific nucleotides, the forward primer for the first product had extra sequence on the 5 ' end that would allow insertion by recombination into a pET21b vector (Table IV). The reverse primer for the first fragment contained extra sequence on the 5' end that was complimentary to the sequence at the beginning of fragment 2.

[149] The forward primer for fragment 2 had additional 5' sequence corresponding to Attorney Docket No: STG-188 Customer No.: 27,495 the end sequence of fragment 1 , as well as containing the same sequence encoding the beginning of fragment 2. In addition to Pfu DNA polymerase sequence, the reverse primer of fragment 2 also contained sequence homologous to the 6X His region of the pET21 b vector which allows for homologous recombination into the vector.

Table IV

JdF3 xιF TTGTTTAACTTTAAGAAGGAGATATACATATGATCCTTGACGTTGATTACATCACCGAGAATGG

J-PchimlntF GGA AAG GCA GAA GAT AAA GAG GAA ATG A AG GAA ACT CAA GAT CCT ATA GAA AAA ATA C

J-PchimlntR GTA TTT TTT CTA TAG GAT CTT GAG TTT CCT TCA TTT CCT CTT TAT CTT CTG CCT TTC C

JDF3xι Rhis CCGGATCTCAGTGGTGGTGGTGGTGGTGCTTCTTCTTCCCCTTCGGCTTCAGC

[150] Amplification reactions were assembled as follows:

[151] Fragment I PCR setup

10 μl 5x Herculase II buffer (Stratagene)

0.4 μl 10OmM dNTP mix (25mM each)

1.2 μl JdF3XiF primer 125μlM

1.2 μl JPchimR primer 125 μpM

0.5 μl exo- JdF-3 DNA polymerase plasmid clone (~50ng/μl)

1.0 μl Herculase II Fusion DNA polymerase (Stratagene)

35.7 μl H20

[152] Fragment 2 PCR setup

10 μl 5x Herculase II buffer (Stratagene)

0.4 μl 10OmM dNTP mix (25mM each)

1.2 μl JPchimF primer 125μlM

1.2 μl PfuXiR primer 125μlM

0.5 μl exo-Pfu DNA polymerase plasmid clone (50ng/μl)

1.0 μl Herculase II Fusion DNA polymerase (Stratagene)

35.7 μl H2O Attorney Docket No: STG-188 Customer No.: 27,495

[153] Amplification conditions: 1 cycle 95°C 1 min 30 cycles 95°C 45 sec 500C 45 sec 1 cycle 72°C 1 min

[154] After amplification, Dpn I restriction enzyme was added to the reactions to destroy the parental plasmid DNA template. The products were purified with the StrataPrep PCR kit per manufacturer's instructions.

[155] Splice Overlap setup

5 μl 1Ox cPfu Buffer

0 .4 μl 10OmM dNTP (25mM each)

5 μl pure fragment 1

5 μl pure fragment 2

1 μl Pfu Turbo DNA polymerase (Stratagene)

33.6 μl H2O

[156] Amplification conditions:

Lc ycle

95C 5C 1 min

15 cycles

95C 5C 45 sec

50c 5C 45 sec

72C 5C 1 min, 50 sec Attorney Docket No: STG-188 Customer No.: 27,495

[157] One microliter of the splice overlap reaction was used as template in the reaction below:

5 μl 10 cPfu Buffer

0.4 μl 10OmM dNTP (25mM each)

1.2 μl JdF3XiF primer 125 μl M

1.2 μl PfUXiR primer 125 μl M

1 μl overlap reaction

1 μl Pfu Turbo DNA polymerase (Stratagene)

40.2 μl H20

[158] Amplification conditions: 1 cycle 95°C 1 min 30 cycles 95°C 45 sec 500C 45 sec 72°C 3 min

[159] The PCR products were gel purified and cloned into a pET21 vector which had been prepared using the Xi-Clone High Speed Cloning Kit (Genlantis) per manufacturer's instructions (as described under Vector Preparation in Example 3). The clones were transformed into XLlO-GoId ultracompetent cells(Stratagene) and incubated at 300C for approximately 20 hours. Colonies were screened by PCR for an insert of the correct size. Positive colonies were cultured and DNA purification was performed. The plasmid insert was sequenced to confirm the split region and the 5' and 3' insertion sites. [160] Verified plasmids were transformed into BL21 -CodonPlus (DE3)-RIL (or RILP) competent cells per manufacturer's instructions (Stratagene) and expressed and purified as described in other sections. Attorney Docket No: STG-188 Customer No.: 27,495

[161] Example 6: Other split polymerases comprising mutations [162] The 4Cl 1 Pfu split polymerase and the JdF-3 Z2 polymerase provide two substantially different types of splits. The 4Cl 1 split is a "seamless split" in which no amino acids are introduced into or removed at the split site, and the sequence of amino acids near the split remains unchanged as compared to the wild type Pfu. The JdF-3 Z2 split polymerase includes a 10 amino acid insertion on the end of fragment I, the N- terminal fragment, including a duplication of the tripeptide of amino acids 467-469. This demonstrates a tolerance for insertions at the split site.

[163] A number of additional amino acid mutations were investigated in the context of the 4Cl 1 mutant. The amino acid mutations were remote from the split site, which was after amino acid 467. The Q484R mutation increased utilization of dual labeled nucleotide analogs as compared to the 4Cl 1 mutant without the mutation. A number of other point mutations are known to increase incorporation of non-conventional nucleotide analogs such as positions corresponding to Pfu L409, Y410, P411, R461, K465, Q472, Q484, A486, R488, L490, A491, N492, Y495, and Y497. Such mutations may be useful in increasing the incorporation of non-conventional nucleotides by split polymerases in extension reactions. The three additional mutations, A318T, V604L, and A662V may contribute to the stability of the split polymerase. Other mutations that were subsequently added to 4Cl 1, at D141A/E143A to reduce 3' to 5' exonuclease activity, and addition of an Sso-like DNA binding domain at the C-terminus of the C-terminal fragment, did not disrupt the increased dual labeled nucleotide analog incorporation observed in the initial 4C 11 mutant. This demonstrates that mutations made at sites remote to the split site can have their expected activity without altering the increased dual label nucleotide analog incorporation observed with the split polymerase without further mutations.

[164] Further split polymerases were designed in Pfu and JdF-3 using some of the primers shown in Table V. Some of the split polymerases have been expressed, purified, and tested for polymerase activity and improved non-conventional nucleotide Attorney Docket No: STG-188 Customer No.: 27,495 incorporation (Table VI). Sites of splits and amino acid changes are indicated by the sites in the native polypeptide. For example, the same split found in the Pfu split polymerase 4Cl 1, was also made in an identical site in the JdF-3 polymerase (mutants JdF-3 201 and JdF-3 202 (shown in Fig. 4)).

Table V

Table VI

Attorney Docket No: STG-188 Customer No.: 27,495

All mutants in Table VI are exo-minus and include mutations at amino acids D141A and E143A.

[165] Test for activity in standard PCR for conventional nucleotide incorporation or for analog incorporation was performed using the method in Example 4. Conventional nucleotide incorporation is scored as a "yes" if a product of the expected size is Attorney Docket No: STG-188 Customer No.: 27,495 detectable by ethidium bromide staining of an agarose gel (at least about 1 ng of nucleic acid). Analog incorporation is scored as a "yes" if a product of the expected size is detected prior to ethidium bromide staining of a comparable agarose gel under the appropriate wavelength for detection of the analog. The amount of nucleic acid detected depends on the percent analog used in the reaction, and the amount of product produced. QPCR methods can also be used to detect analog incorporation. [166] It is notable that the splits at amino acid 547, roughly analogous to the natural split Mth archaeal polymerase, inhibit function of Pfu DNA polymerase. [167] Figure 8 shows non-conventional nucleotide incorporation assays for some of the other mutants tested. For each variant, the enzyme was purified from a IL culture after expression from a pET vector in E. coli strain BL21-DE3-CodonPlus (RIL). Purification included the following steps: heat-treatment of the cell lysate at 85°C for 15 minutes; treatment of the heated lysate with 0.15% PEI (polyethyleneimine) and 1 M NaCl to remove nucleic acids; clarification by centrifugation; then column chromatography on Q- Sepharose FF followed by Heparin-Sepharose HP. Enzyme was found in the flow- through fraction for Q-Sepharose and was eluted with a KCl gradient from Heparin- Sepharose. The purified enzymes were analyzed by SDS-PAGE. Variant JDF-3 Z2 was comprised of predominantly two peptide fragments, as expected. Other, non-split variants were comprised of single fragments.

[168] PCR activity was measured for JDF-3 variants using a 962bp Lambda DNA target. The reaction was carried out in IX Taq PCR reaction Buffer in a 25 μl reaction containing 5ng Lambda DNA, 0.4 μM each primer (Forward: 5'- ATCAGAAACGAACGCATCATCAAGT, Reverse: 5'-

GCCTCGCATATCAGGAAGCAC), 200 μM each dGTP, dATP, TTP, and the indicated amounts of dCTP, FCD, and/or RCD (5-aminoallyl-(5-ROX)-2'-deoxycytidine-5'- triphoso-N6-(6-aminohexyl)-dabcyl). Cycling conditions were: 93°C, lmin; 30 cycles of 93°C, lmin, 58°C,50sec, 72°C, lmin; then 72°C, lOmin. Samples (5 μl or 75 ng of each reaction were run on a 1% agarose gel. The gel was photographed first in the absence of ethidium bromide with a Green filter (A) to visualize incorporated fluorescein (FCD) or Attorney Docket No: STG-188 Customer No.: 27,495 with an ethidium bromide filter (B) to visualize incorporated ROX (RCD). Then, the gel was stained with ethidium bromide and photographed again with the ethidium bromide filter (C). Samples with the "a" designation comprised 25 μM FCD + 25 μM dCTP in the reaction mix and samples with a "b" designation comprised 5 μM RCD + 45 μM dCTP. The samples loaded were exo-minus JdF-3 (samples designated as "1"), exo- minus JdF-3, L408H, A490Y ("2"), exo-minus JdF-3 Z2 ("3"), exo-minus Pfu 4Cl 1 ("4"), and exo-minus Pfu, L409H, A491Y ("5"). As stated previously, the exo-minus notation means that additional mutations D 14 IA and E 143 A are present in the polymerase.

[169] Both FAM (FAM-dCTP-Dabcyl uptake) incorporation (Panel A) and ROX incorporation (Panel B) were increased with the JdF-3 Z2 (lane 3 of each gel in Fig. 8), JdF-3 L408H and A490Y (lane 2), and Pfu L409H and A491 Y (lane 5) mutants as compared to wild type JdF-3 (lane 1).

[170] Figure 9 shows the amino acid sequence of the 4Cl 1 DNA polymerase with the Q484R mutation, comprising both the amino fragment (Panel A) and the carboxyl fragment (Panel B). The sequence has the D141A/E143A mutations (exo minus). The changed amino acids are shown slightly above the rest of the sequence. The corresponding nucleic acid sequence (SEQ ID NO: 80) is shown in the sequence listing below.

[171] Figure 10 depicts the results from a PCR amplification assay using the Pfu double mutant. This is a split Pfu mutant with the exo-minus mutations and with an additional Q484R mutation (different from the exo-minus Pfu 4Cl 1 mutant described in Example 2). Amplification was performed with a dCTP analog labeled with dabcyl at the gamma phosphate. The nucleotide pool used was 200μM dATP, dGTP and TTP. The total pool of dCTP was 50μM and the percentage of dabcyl-dCTP in that pool varied from zero to 100 percent as indicated on the figure. The double mutant was compared to Exo- minus cPfu, a non-split polymerase that was exonuclease minus (comprised mutations D 14 IA and E 143A). As can be seen from Figure 10, the activity of the double mutant and the non-split enzyme are substantially the same at lower concentrations of the dCTP analog Attorney Docket No: STG-188 Customer No.: 27,495

(up to 75 μM). However, at higher ratios dCTP analog (at 75 and 90 μM), the non-split polymerase loses activity, but the double mutant configuration particularly improves the uptake of dabcyl dCTP modified at the gamma phosphate. Without being limited to any mechanism, this perhaps is a result of a loosening of the nucleotide binding pocket when the finger folds over the incoming nucleotide.

[172] Figure 11 depicts the results from PCR amplification of a plasmid template with dCTP labeled with the nucleotide analog FCD. End point PCR (plasmid template in Ix cPfu buffer; Stratagene) using 200μM each dATP, dGTP and TTP, and a total dCTP (dCTP+FCD) pool of 50μM was used in the reactions. The percentage of FCD in the dCTP pool varied from zero (100% dCTP:0% FCD) to one hundred percent (0% dCTP: 100% FCD) as indicated in Figure 11. The 4Cl 1 mutant with the exo minus mutations (D141A and E143A) was compared to the non-split Pfu enzyme with the exo minus mutations (D141A and E143A). Electrophoresis of the PCR products on an agarose or acrylamide gel in the absence of ethidium bromide allowed the resultant FAM-labeled DNA products to be visualized with ultraviolet excitation and a green filter (Panel A). The gel was subsequently stained with ethidium bromide and visualized with an orange filter to reveal all DNA molecules (Panel B). As shown in the figure, the wild type (non-split) exo minus Pfu enzyme was unable to incorporate the FCD analog, while the split Pfu Q484R double mutant showed an optimal uptake of FAM-dCMP at 50-75% FCD. This experiment shows that the split polymerases of the present invention are not limited by the kind of nucleotide analog that can be used, but that the invention works for different types of modified nucleotides.

[173] Example 7: Refolding of 4Cl 1 split polymerase after separate expression of each fragment

[174] In this Example, the N-terminal and C-terminal fragments of the 4Cl 1 split polymerase were expressed and purified separately as in Example 3. However, in this case, the fragments were also solubilized and refolded (Figure 12). Each was expressed from a pET vector in E. coli strain BL21-DE3-CodonPlus RIL in a 2L culture. The Attorney Docket No: STG-188 Customer No.: 27,495 insoluble pellets (i.e. the inclusion bodies) after cell breakage were washed in turn with buffer containing 0.15% sodium deoxycholate, 1% Triton X-IOO, and then with buffer alone (40 mM Tris pH 7.5, 10 mM 2-mercaptoethanol). The N-terminal and C-terminal fragment pellets were solubilized with 4M or 5M guanidine HCl, respectively. SDS- PAGE samples were prepared from the guanidine HCl-containing preparations by TCA precipitation.

[175] The partially-purified fragments were visualized by SDS-PAGE (Panels A and B). Results showed that both the N-terminal fragment (Panel A) and the C-terminal fragment (Panel B) were expressed, although in different amounts. The N-terminal fragment was approximately 50% soluble and made in large amounts. The C-terminal fragment, however, was much less abundant, and completely insoluble when expressed on its own. Based on the band intensity of the fragments compared to the intensity of staining for the 4Cl 1 control, it was estimated that the N-terminal fragment preparation was approximately 5 -fold more concentrated than the C-terminal fragment. The C-terminal fragment preparation was also less pure than the N-terminal fragment, possibly due to the initially lower yield of expressed C-terminal fragment.

[176] For refolding experiments, the insoluble fraction of each sample was purified. Refolding experiments were performed by simply mixing the two solubilized fragments in an approximately 1 :1 stoichiometric ratio (i.e. 5:1 volume of C-terminal: N-terminal). Controls included reactions with each fragment alone. Refolding was achieved by simply dialyzing the mixture against 200 volumes of buffer (40 mM Tris, pH 7.5, ImM EDTA, 100 mM KCl, 10% glycerol) containing no guanidine HCL and no 2-mercaptoethanol. The dialysis buffer was changed twice over a ~24hr period. A multi-step dialysis was also tried where the guanidine HCl concentration was reduced in steps (2M, IM, OM), but produced nearly identical results as the single step method. After recovery of the samples from dialysis, insoluble material was removed by centrifugation. Protein in the samples was quantitated by Bradford assay.

[177] Refolded samples were analyzed by SDS-PAGE (Panel C). The samples loaded were solubilized N-terminal fragment (N-term), solubilized C-terminal fragment (C- Attorney Docket No: STG-188 Customer No.: 27,495 term), purified 4Cl 1 control (4Cl 1), complete refolding reaction comprising N-terminal and C-terminal fragments (1), control refolding reaction comprising N-terminal fragment only (2), and control refolding reaction comprising C-terminal fragment only (3). The Bradford protein assays showed recovered soluble protein of 1.0 mg/ml for sample 1, 1.1 mg/ml for sample 2, and < 0.1 mg/ml for sample 3. Interestingly, the N-terminal fragment largely refolded to a soluble state on its own. It showed slightly more recovered soluble protein than for the reaction with both fragments, possibly indicating that the very insoluble C-terminal fragment co-precipitated with a portion of the N-terminal fragment during refolding.

[178] Refolded proteins were also analyzed by PCR assays to demonstrate successful refolding (Panel D). The samples loaded were Pfu Ultra II polymerase as a control (Ultra II), refolding reaction comprising N-terminal and C-terminal fragments (#1), refolding reaction comprising N-terminal fragment only (#2), and refolding reaction comprising C- terminal fragment only (#3). The indicated volumes of each refolding reaction (and ng amounts of Pfu Ultra II polymerase for the controls) were assayed by PCR (0.9 kb human alpha- 1 -antitrypsin target) in 50 μl reactions consisting of IX Pfu Ultra II reaction buffer, 100 ng human genomic DNA, 100 ng each primer (Forward: 5'- GAGGAGAGCAGGAAAGGTGGAAC; Reverse:

5 'GAGGTACAGGGTTGAGGCTAGTG), and 250 μM each dGTP, dATP, dCTP, TTP. Cycling conditions: 1 cycle of 95°C, 2min; 30 cycles of 95°C, 30sec, 58°C, 30sec, 72°C, 15sec, then 1 cycle of 72°C, lOmin. 10 μl of each PCR reaction was analyzed on a 1% agarose gel. All reactions were run in duplicate. Results clearly show that PCR assays were only successful when both the N-terminal and C-terminal fragments were present. PCR amplification products were only seen in lanes containing sample #1 and the size of the products correlated to the size of the products seen in the control lanes (Ultra II). [179] A refolded sample was also subjected to chromatographic purification (Panel E) to demonstrate its integrity and to demonstrate that it behaved similarly to the enzyme purified from a bicistronic expression vector. A refolded sample acquired as described above using both N-terminal and C-terminal fragments was further purified using a Attorney Docket No: STG-188 Customer No.: 27,495 standard method for Pfu polymerases. This included Q-Sepharose column chromatography where Pfu polymerase passes through the column without binding, followed by SP-Sepharose chromatography where Pfu polymerase binds and is subsequently eluted with a KCl gradient. The purified samples were analyzed by SDS- PAGE. The samples loaded were the initial refolded sample (which was the Q-Sepharose load), the Q-Sepharose flow thru, the SP-Sepharose flow thru, and SP-Sepharose gradient fractions (Cl 1, DI l, D8, D5, and D2). Results indicated that the refolded polymerase was successfully bound by SP-Sepharose and eluted in a purified form by the gradient. If the split polymerase is expressed from a bicistronic expression vector, the polypeptide fragments do not form inclusion bodies and therefore, do not need to be solubilized and refolded. Therefore, a preferred embodiment of the methods of the invention comprises production of a split polymerase of the invention using a polycistronic expression system to avoid the steps of solubilization from inclusion bodies and refolding of the fragments into a functional split polymerase. Expression of a split polymerase from a polycistronic expression system has advantages of ease of production, cost-effectiveness, time savings, etc.

[180] It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[181] For example, in some cases the fingertip region may be comprised of an additional amino acid. In such cases, the consensus sequence for the split site will more closely resemble:

GXXXXBLXXLBXXRX-BKXXMXXJX(1- 2)DXXOZXBLDXRQZABKBBANXUYGYXXX Attorney Docket No: STG-188 Customer No.: 27,495 in which the lettering has the same meaning as described elsewhere, except that X(I-2) means that either one or two amino acids can be in that region.

Attorney Docket No: STG-188 Customer No.: 27,495

CLAIMS

1. A non-natural split polymerase.

2. The split polymerase of claim 1, wherein the split polymerase is a thermostable DNA polymerase.

3. The split polymerase of claim 1, wherein the split polymerase is a Family B DNA polymerase.

4. The split polymerase of claim 1, wherein the polymerase is a DNA polymerase from a Pyrococcus or Thermococcus species.

5. The split polymerase of claim 4, which is a JdF-3 DNA polymerase comprising SEQ ID NO:91 and SEQ ID NO:92, with or without a K464A mutation, or a JdF-3 DNA polymerase comprising SEQ ID NO:97 and SEQ ID NO:98.

6. The split polymerase of claim 4, which is a Pfu DNA polymerase comprising SEQ ID NO:94 and SEQ ID NO:95.

7. The split polymerase of claim 1 , wherein the split is within the fingers domain of the polymerase.

8. The split polymerase of claim 1, wherein the split polymerase increases incorporation of non-natural nucleotides or increases utilization of modified primer templates relative to the non-split polymerase.

9. The split polymerase of claim 1, wherein the polymerase comprises a motif

- 80 - Attorney Docket No: STG-188 Customer No.: 27,495 having the sequence RXXXK(X)nQXXXXKXXXNSXYG (SEQ ID NO:4), where n is any number of amino acids between and including 15 and 80, and wherein the split occurs within this sequence.

10. The split polymerase of claim 1, wherein the split is located in the fingers domain and has at least 70% identity to amino acids 448 to 500 of the Pfu polymerase.

11. The split polymerase of claim 1 , wherein the split polymerase comprises one or more mutations that improve incorporation of a non-natural nucleotide into a nucleic acid polymerized by the split polymerase, that reduce 3 '-5' exonuclease activity, that reduce

5 '-3' exonuclease activity, that reduce uracil detection activity, that increase stability of the split polymerase as compared to a corresponding non-split polymerase, or that incorporate a DNA binding domain.

12. The split polymerase of claim 1, wherein the amino acid sequence of the polymerase comprises a mutation at an amino acid corresponding to one or more of the following amino acids in the Pfu DNA polymerase: L409, Y410, P411, R461, K465, Q472, Q484, A486, R488, L490, A 491, N492, Y495, and Y497.

13. The split polymerase of claim 12, wherein the amino acid mutation corresponds to one or more of the following Pfu mutations: L409H, Y410V, P41 IL, R461A/N, K465A/N, Q472H, Q484R/K, A486T, R488A, L490Y, A491Y, N492A, Y495S, Y497A/L.

14. The split polymerase of claim 1, wherein the amino acid sequence of the polymerase comprises, with respect to a corresponding Pfu DNA polymerase sequence, a double mutation D141A/E143A, a single mutation V93R, or both, which reduce 3' to 5' exonuclease activity or reduce sensitivity to uracil as compared to the same polymerase without the mutations.

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15. The split polymerase of claim 1, wherein the amino acid sequence of the polymerase comprises a mutation at an amino acid corresponding to one or more of the following amino acids in the Pfu DNA polymerase: A318, V604, A662, wherein the mutations increase stability of the split polymerase as compared to the polymerase without the mutations.

16. The split polymerase of claim 1 which further comprises a C-terminal fusion with a DNA binding protein.

17. A composition comprising the split polymerase of claim 1 and at least one other substance required for a nucleic acid polymerization reaction.

18. A nucleic acid encoding a non-natural split polymerase or a functional fragment thereof.

19. The nucleic acid of claim 18, wherein the nucleic acid encodes a polymerase that is split in the fingers domain of the polymerase.

20. The nucleic acid of claim 18, wherein the nucleic acid comprises at least one mutation that improves incorporation of a non-natural nucleotide, reduces 3 '-5' exonuclease activity, reduces 5 '-3' exonuclease activity, reduces uracil detection activity, increases stability, or that incorporates a DNA binding site when expressed as a split polymerase, wherein the differences are seen between the encoded split polymerase and a non-split wild-type polymerase from which it is derived.

21. The nucleic acid of claim 18, wherein the nucleic acid further comprises at least one mutation whereby a split polymerase encoded by the nucleic acid comprises a mutation in at least one amino acid corresponding to the following amino acids of Pfu

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DNA polymerase: L409, Y410, P411, R461, K465, Q472, Q484, A486, R488, L490, A 491, N492, Y495, Y497, D141, E143, V93, A318, V604, and A662.

22. The nucleic acid of claim 18, which comprises a coding region encoding SEQ ID NO:91 and a separate coding region encoding SEQ ID NO:92, or which comprises a coding region encoding SEQ ID NO:97 and a separate coding region encoding SEQ ID NO:98.

23. The nucleic acid of claim 18, which comprises a coding region encoding SEQ ID NO: 94 and a separate coding region encoding SEQ ID NO: 95.

24. A composition comprising the nucleic acid of claim 18 and at least one other substance required to replicate or amplify the nucleic acid.

25. A method for DNA synthesis comprising: combining a non-natural split DNA polymerase with a nucleic acid template; and providing conditions that permit DNA synthesis.

26. The method of claim 25, wherein the non-natural split polymerase comprises a split polymerase with increased ability to incorporate non-natural nucleotides or increased ability to utilize modified primer templates relative to a non-split polymerase derived from the same sequence.

27. The method of claim 25, wherein the method of DNA synthesis is a method of DNA sequencing, quantitative PCR (QPCR), DNA labeling, or a combination thereof.

28. The method of claim 25, wherein the non-natural split polymerase comprises SEQ ID NO:91 and SEQ ID NO:92, with or without a K464A mutation, or SEQ ID NO:94 and SEQ ID NO:95, or SEQ ID NO:97 and SEQ ID NO:98.

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29. A kit comprising a non-natural split polymerase.

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