| 摘要 |
The invention relates to a method for amplifying a target nucleic acid sequence tn-tC-tV-tC′-tn′ comprising a first amplification using a first primer pair with sequence ma-K-pC and ma-K′-pC′, and a subsequent second amplification using a second primer pair with sequence aL-aP-aK and aL′-aP′-aK′, wherein pC is the same sequence as sequence element tC. pC and pC′ are 8 to 40 nucleotides in length, K comprises a 3′-terminal sequence k1-k2-s, s is a mismatch sequences preventing PCR bias, ak is the same sequence as sequence element k1, aP-aK hybridize to a contiguous sequence on sequence element ma-K, k1 is 2 to 9 nucleotides in length, aL and aL′ can be any sequence, and tV is a variable region within said target nucleic acid sequence. The invention further relates to collections of primer sets for use in the method of the invention. |
| 主权项 |
1. A method for amplifying a target nucleic acid sequence tC-tV-tC′ comprised within a sequence tract tn-tC-tV-tC′-tn′, said method comprising conducting a plurality of amplification reactions, each reaction comprising
a first amplification step, whereby said target nucleic acid sequence is amplified using
i. a left (forward) initial PCR primer having a sequence ma-K-pC andii. a right (reverse) initial primer having a sequence and ma-K′-pC′, yielding a first amplificate, a second amplification step, whereby said first amplificate is amplified using
iii. a left (forward) adaptor PCR primer having a sequence aL-aP-aK andiv. a right (reverse) adaptor PCR primer having a sequence aL′-aP′-aK′, yielding a second amplificate, wherein tV is a variable region within said target nucleic acid sequence, pC is the same sequence as sequence element tC and pC′ is the reverse complimentary sequence to tC′, K comprises a sequence element k1 and a 3′-terminal sequence element s, and K′ comprises a sequence element k1′ and a 3′-terminal sequence element s′, wherein
k1 and k1′ each independently from one another are a sequence 2 to 9 nucleotides in length,s and s′ are mismatch sequences selected not to form a continuous hybrid sequence with sequence element tn and tn′, and s and s′ are each independently 1, 2, 3, 4 or 5 nucleotides in length, ak is the same sequence as sequence element k1 and aK′ is the same sequence as sequence element k1′, aP-aK hybridizes to a contiguous sequence on ma-K and aP′-aK′ hybridizes to a contiguous sequence on ma-K′, pC, pC′, ma-K and ma-K′ each independently from one another are a sequence 10 to 40 nucleotides in length, and aL and aL′ independently from one another can be any sequence, and wherein a particular set of primers for each one of said plurality of amplification reactions is provided, wherein in each of these sets of primers k1 of said particular set is different from any other k1 in any of the other sets and/or k1′ of said particular set is different from any other k1′ in any of the other sets. |
