Method for reducing primer-dimer amplification
The present invention reduces primer-dimer amplification in a multiplex polymerase chain reaction (PCR). When a first forward primer (F1) and a second reverse primer (R2) have a complementary region at their 3′ends, primer dimers may form. The present method uses a primer comprising a 5′-end partial sequence or a full sequence of a first forward primer (F1^) in between a first tag (t1) and R2 to reduce the primer-dimer (F1_R2) amplification.
1. A method for reducing primer-dimer amplification in a multiplex polymerase chain reaction (PCR), comprising the steps of:
(a) obtaining a first nucleic acid sequence comprising a first tag (t1) and a first forward primer (F1) complementary to a first target nucleic acid fragment,
(b) obtaining a second nucleic acid sequence comprising a second tag (t2) and a first reverse primer (R1) complementary to the first target nucleic acid fragment,
(c) obtaining a third nucleic acid sequence comprising a third tag (t3) and a second forward primer (F2) complementary to a second target nucleic acid fragment,
(d) obtaining a fourth nucleic acid sequence comprising the first tag (t1), a second reverse primer (R2) complementary to the second nucleic acid fragment, and a 5′-end partial sequence (F1^) or a full sequence of the first forward primer (F1) in between the first tag (t1) and the second reverse primer (R2), wherein the first forward primer (F1) and the second reverse primer (R2) have a complementary region at their 3′ends, F1^ has 3-30 nucleotides or 40-90% of the 5′-end partial F1 sequence,
(e) mixing the first and the second target nucleic acid fragments, the first, the second, the third, and the fourth nucleic acid sequences, and an effective amount of reagents necessary for performing a polymerase chain reaction (PCR); and
(f) performing PCR.
2. The method according to claim 1 , wherein step (f) comprises:
(f1) activating DNA polymerase and denaturing DNAs in the mixture of (e),
(f2) cycling the mixture of (f1) through denaturing, annealing and primer extension steps of PCR multiple times to obtain amplification products.
3. The method according to claim 1 , wherein step (f) comprises:
(f-i) activating DNA polymerase and denaturing DNAs in the mixture of (e),
(f-ii) cycling the mixture of (f-i) through denaturing, annealing and primer extension steps of PCR at least two times, and
(f-iii) cycling the mixture of (f-ii) through denaturing, annealing and primer extension steps of PCR multiple times at an annealing temperature higher than that in step (f-ii) to obtain amplification products.
4. The method according to claim 3 , wherein the annealing temperature in step (f-iii) is 4-35° C. higher than the annealing temperature in step (f-ii).
5. The method according to claim 1 , wherein tags t3 and t2 have the same sequence.
6. The method according to claim 1 , wherein the sequences of both tags t3 and t2 are different from the sequence of tag t1.
7. The method according to claim 1 , wherein F1^ has 3-30 nucleotides of the 5′-end partial F1 sequence.
8. The method according to claim 4 , wherein F1^ has 3-30 nucleotides of the 5′-end partial F1 sequence.
9. The method according to claim 1 , wherein F1^ has 40-90% of the 5′-end partial F1 sequence.
10. The method according to claim 4 , wherein F1^ has 40-90% of the 5′-end partial F1 sequence.
11. The method according to claim 1 , wherein F1, F2, R1, and R2 are gene-specific primers.