WO 2008/092213 A1 - Generation Of Nucleic Acid Molecules - The Lens

Generation Of Nucleic Acid Molecules

Published: Aug 7, 2008
Earliest Priority: Feb 02 2007
Family: 19
Cited Works: 32
Cited by: 4
Cites: 19
Additional Info: Cited Works Full text Published

Abstract

The present invention relates generally to methods for generating single stranded nucleic acid molecules following enhanced solid phase polynucleotide amplification. The present invention employs an amplification reaction using primers with differential priming properties at particular annealing conditions or an immobilised primer nested between two aqueous phase primers. Thus, by primer design, solid support primer participation is enhanced relative to aqueous phase primers. The subject invention further provides methods for labelling solid matrices with single and double stranded nucleic acid molecules. Kits for generating single stranded nucleic acid molecules and for conducting amplification reactions also form part of the present invention. The present invention further provides amplification systems for the generation of single stranded nucleic acid molecules optionally labelled with a reporter molecule and their use inter alia as labels, primers and probes.

Claims

- 48 -
A method for generating a single stranded polynucleotide in a reaction vessel, said method comprising subjecting a target double stranded polynucleotide or its single stranded derivative to exponential amplification by contacting a solid matrix having a primer bound to said solid matrix via linker means with a sample comprising at least one nucleic acid molecule wherein the immobilized primer is selected from the list consisting of:
(i) a primer sharing sequence identity to an aqueous phase primer but having a different melting temperature relative to the immobilized primer; and
(ii) a primer nested between two aqueous phase primers;
under reaction conditions which effect elongation of said immobilized primer to facilitate the generation of double stranded polynucleotide immobilized to the solid matrix and then subjecting the immobilized polynucleotide to denaturing conditions to generate immobilized single stranded polynucleotides and a liquid phase single stranded polynucleotide.
The method of Claim 1 further comprising a phase separation step to isolate immobilized or aqueous phase single stranded polynucleotide.
A method for solid phase amplification of nucleic acid molecules which comprises contacting a solid support having a primer bound to said solid support via linker means with a sample comprising at least one nucleic acid molecule wherein the immobilized primer is selected from the list consisting of:
(i) a primer sharing sequence identity to an aqueous phase primer but having a different melting temperature relative to the immobilized primer; and - 49 -
(ii) a primer nested between two aqueous phase primers;
under reaction conditions which effect elongation of said immobilized primer.
The method of Claim 1 or 3 wherein the different melting temperatures are due to one or more of different annealing temperatures, extent of mismatch between primer and target complementary polynucleotide and/or the presence of extraneous sequences.
The method of Claim 4 wherein one or both of the primers comprise a head or heel nucleotide sequence which may or may not be related by homology to the target complementary polynucleotide thereby providing a higher melting temperature compared to the other primer.
The method of Claim 1 or 3 or 4 or 5 wherein the target to be amplified comprises either a recessed 5' end or a blunt end which is incubated with a 5' to 31 exonuclease to generate a single stranded polynucleotide template.
A method for labeling a solid matrix with a single stranded polynucleotide, said method comprising subjecting a target double stranded polynucleotide or its single stranded derivative to exponential amplification using forward and reverse primers having similar annealing properties at a first set of annealing conditions but differential annealing properties at a second set of annealing conditions, contacting the amplification product of the amplification with a solid matrix or composition of solid matrices having immobilized thereon at least one of the primers which is capable of annealing to a strand of the amplification product under the second set of annealing conditions and altering the annealing conditions to the second set of conditions to thereby facilitate amplification based on a single primer to generate a single stranded polynucleotide immobilized to said solid matrix.
A method for labeling a solid matrix which comprises contacting a solid matrix having a primer bound to said solid matrix via linker means with a sample comprising at - 50 -
least one nucleic acid molecule wherein the immobilized primer is selected from the list consisting of:
(i) a primer sharing sequence identity to an aqueous phase primer but having a different melting temperature relative to the immobilized primer; and
(ii) a primer nested between two aqueous phase primers;
under reaction conditions which effect elongation of said immobilized primer.
The method of Claim 7 or 8 wherein the different annealing conditions or different melting temperatures is due to one or more of different annealing temperatures, extent of mismatch between primer and target complementary polynucleotide and/or the presence of extraneous sequences.
The method of Claim 9 wherein one or both of the primers comprise a head or heel nucleotide sequence which may or may not be related by homology to the target complementary polynucleotide thereby providing a higher melting temperature compared to the other primer.
The method of Claim 7 or 8 or 9 or 10 wherein the target to be amplified comprises either a recessed 5' end or a blunt end which is incubated with a 5' to 31 exonuclease to generate a single stranded polynucleotide template.
The method of Claim 7 or 8 or 9 or 10 further comprising after subjecting the reaction to the second set of annealing conditions, altering back to a permissive set of annealing conditions to facilitate generation of a complementary strand on the immobilized single stranded polynucleotide to form a duplex polynucleotide immobilized to a solid matrix.
The method of any one of Claims 1 to 12 wherein the polynucleotide is DNA. - 51 -
A method of generating ssDNA in a reaction vessel, said method comprising conducting an amplification reaction of a target immobilized ssDNA template from a dsDNAor mRNA target in the reaction vessel using a pair of forward and reverse primers having similar (balanced) annealing primers at a first set of annealing conditions (mutually permissive conditions) but differential annealing properties at a second set of annealing conditions (differentially permissive conditions) wherein the amplification is permitted to proceed under the mutually permissive annealing conditions; altering the conditions to differentially permissive conditions to unbalance the primers thereby facilitating linear amplification substantially in the presence of only a single primer to generate ssDNA product; and inactivating any polymerase activity to substantially reduce or prevent dsDNA formation.
A method for generating a solid matrix or composition of solid matrices labeled with double stranded polynucleotides, said method comprising subjecting a target double stranded polynucleotide or its single stranded derivative to exponential amplification by contacting a solid matrix having a primer bound to said solid matrix via linker means with a sample comprising at least one nucleic acid molecule wherein the immobilized primer is selected from the list consisting of:
(i) a primer sharing sequence identity to an aqueous phase primer but having a different melting temperature relative to the immobilized primer; and
(ii) a primer nested between two aqueous phase primers;
under reaction conditions which effect elongation of said immobilized primer.
The method of Claim 14 or 15 wherein the target to be amplified comprises either a recessed 5' end or a blunt end which is incubated with a 5' to 3' exonuclease to generate a single stranded polynucleotide template. - 52 -
The method of Claim 6 or 11 or 16 wherein the exonuclease is incubated together with reagents required for isothermal amplication.