Patent ID: 11913069
Assignee: PILLAR BIOSCIENCES INC.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 10:
11. A method for selectively amplifying target nucleic acid fragments having an overlapping region, comprising the steps of:
(a) obtaining a first nucleic acid sequence comprising a first tag (t2) 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 (t1) and a first reverse primer (R1) complementary to the first target nucleic acid fragment,
(c) obtaining a third nucleic acid sequence comprising the second tag (t1) and a second forward primer (F2) complementary to a second target nucleic acid fragment,
(d) obtaining a fourth nucleic acid sequence comprising a third tag (t3), and a second reverse primer (R2) complementary to the second nucleic acid fragment, wherein the first and the second target nucleic acid fragments have an overlapping region,
(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);
cycling the mixture of (e) through denaturing, annealing and primer extension steps of PCR for at least two times, and
(g) cycling the mixture of (f) through denaturing, annealing and primer extension steps of PCR at an annealing temperature 2-35° C. higher than that in step (f) to obtain amplification products, wherein the third nucleic acid sequence further comprises a 5′-end partial sequence of the first reverse primer (R1{circumflex over ( )}), in between the second tag (t1) and the second forward primer (F2).