Strand-invasion based DNA amplification method
Methods for amplification of a target nucleic acid sequence comprising strand invasion are provided in which strand invasion occurs both at upstream and downstream regions of the target nucleic acid sequence. Further provided are kits and compositions suitable for use in such methods. The methods may comprise amplifying a target nucleic acid sequence comprising a region of unknown sequence, or determining the sequence of a target nucleic acid comprising a region of unknown sequence.
1. A method for amplification of a nucleic acid sequence of unknown sequence, wherein said nucleic acid sequence is flanked by an upstream binding region comprising a first adaptor sequence and a downstream binding region comprising a second adaptor sequence, the method comprising contacting a nucleic acid comprising said nucleic acid sequence, under appropriate conditions, with:
(a) a first strand invasion oligonucleotide having a non-extendible 3′ termini;
(b) a second strand invasion oligonucleotide having a non-extendible 3′ termini; and
(c) primers comprising an upstream primer and a downstream primer, wherein said primers are capable of amplifying the nucleic acid sequence,
wherein said first strand invasion oligonucleotide binds to said first adaptor sequence and said second strand invasion oligonucleotide binds to said second adaptor sequence, thereby rendering the upstream binding region and the downstream binding region single-stranded to allow binding of said primers, wherein the appropriate conditions are isothermal conditions and include the presence of a recombinase, wherein the method comprises strand invasion at two sites through binding of said first strand invasion oligonucleotide to said first adaptor sequence and said second strand invasion oligonucleotide to said second adaptor sequence, wherein said upstream and downstream binding regions are present in opposing strands of said nucleic acid, and wherein said first strand invasion oligonucleotide and said second strand invasion oligonucleotide are configured to bind to said nucleic acid in an antiparallel orientation.
2. The method according to claim 1 , wherein the upstream and downstream binding regions are present in the same strand of the nucleic acid.
3. The method according to claim 1 , wherein the upstream and downstream binding regions are present in opposing strands of the nucleic acid with their 3′ends directed towards each other.
4. The method according to claim 3 , wherein said first invasion oligonucleotide and said second strand invasion oligonucleotide are configured to bind to the opposing strands of the nucleic acid with their 3′ends directed away from each other.
5. The method according to claim 1 wherein the upstream binding region and/or the downstream binding region of the nucleic acid does not overlap with binding region for the respective upstream or downstream primer.
6. A method of amplifying a target nucleic acid sequence of unknown sequence comprising creating a nucleic acid sequence comprising upstream and downstream strand invasion oligonucleotide binding regions flanking said target nucleic acid sequence of unknown sequence, said binding sequences each incorporating an adaptor sequence, and amplifying said target nucleic acid sequence by carrying out the method according to claim 1 .
7. A method of determining the sequence of a target nucleic acid of unknown sequence, comprising creating a nucleic acid sequence comprising upstream and downstream strand invasion oligonucleotide binding regions flanking said target nucleic acid sequence of unknown sequence, said binding sequences each incorporating an adaptor sequence, amplifying said target nucleic acid sequence by carrying out the method according to claim 1 , and determining the sequence of said region of unknown sequence.
8. The method according to claim 7 , wherein said first strand invasion oligonucleotide and said second strand invasion oligonucleotide have identical sequences.
9. The method according to claim 1 , wherein the nucleic acid sequence is greater than 100 nucleotides in length.
10. The method according to claim 1 , wherein said first strand invasion oligonucleotide and said second strand invasion oligonucleotide have different sequences.
11. The method according to claim 1 , wherein the first adaptor sequence and the second adaptor sequence are identical sequences.
12. The method according to claim 1 , wherein the first adaptor sequence and the second adaptor sequence are different sequences.
13. The method according to claim 1 , wherein the upstream primer and the downstream primer have identical sequences.
14. The method according to claim 1 , wherein the upstream primer and the downstream primer have different sequences.