Generation of nucleic acid molecules
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 immobilized 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 labeling 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.
1. A method for solid phase amplification of a polynucleotide molecule sequence in a reaction vessel comprising:
(a) a) combining in said reaction vessel:
i) a target DNA molecule that comprises the polynucleotide molecule sequence,
ii) a pair of aqueous forward and reverse primers that bind to 5′ and 3′ ends of the polynucleotide sequence, respectively, and
iii) a solid support conjugated to a solid support primer, wherein the solid support primer comprises a target-specific sequence that has at least 80% sequence identity to the forward primer and a 3′ sequence extension that extends beyond the sequence of the forward primer, wherein the target-specific sequence hybridizes to the target DNA molecule, and wherein the Tm of the solid support primer is higher than the Tm of the aqueous forward primer; and
(b) amplifying the polynucleotide molecule sequence by polymerase chain reaction, wherein:
i) during a plurality of first thermal cycles, annealing conditions are permissive to the forward primer, the reverse primer and the solid support primer, and
ii) during a plurality of second thermal cycles conducted after said first thermal cycles, annealing steps have a raised temperature compared to the first thermal cycles, such that, during the second thermal cycles, the solid support primer anneals to target efficiently, but the aqueous forward primer does not, wherein solid phase amplification of the polynucleotide molecule sequence is favored during the second thermal cycles.
2. The method of claim 1 wherein an amplified polynucleotide molecule is immobilized to the solid support, which is then subjected to denaturing conditions to generate immobilized single stranded polynucleotide molecules and aqueous phase single stranded polynucleotide molecules.
3. The method of claim 1 , wherein amplification results in generation of a single-stranded polynucleotide, further comprising isolating the single stranded polynucleotide through phase separation.
4. The method of claim 1 , wherein amplification results in generation of a single-stranded polynucleotide, and wherein the single stranded polynucleotide is immobilized to the solid support and comprises a label capable of generating a detectable signal.
5. The method of claim 1 wherein the polynucleotide 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.
6. The method of claim 1 wherein the solid support primer is conjugated to the solid support by a linking sequence.
7. The method of claim 1 , wherein the solid support is a micro sphere or a microbead.
8. A method of detecting and distinguishing analytes from multiple genomic sources comprising:
amplifying different polynucleotides from the multiple genomic sources, wherein each of the different polynucleotides are amplified by the method of claim 1 , and wherein each of the different polynucleotides are amplified by a different pair of aqueous forward and reverse primers, as recited in claim 1 , and by a different solid support primer, as recited in claim 1 , wherein the different solid support primers are bound to different, identifiable micro spheres or microbeads; and
detecting and distinguishing each of the amplified different polynucleotides on each of the different identifiable micro spheres or a microbeads.
9. The method of claim 8 , wherein the different identifiable microspheres or a microbeads are identifiable on the basis of the microspheres or a microbeads having different diameters.