US 11,913,069 B2
Selective amplification of overlapping amplicons
Zhaohui Wang, Southborough, MA (US); and Gang Song, Newton, MA (US)
Assigned to PILLAR BIOSCIENCES INC., Natick, MA (US)
Filed by Pillar Biosciences Inc., Natick, MA (US)
Filed on Aug. 26, 2021, as Appl. No. 17/458,162.
Application 17/458,162 is a continuation of application No. 16/290,736, filed on Mar. 1, 2019, granted, now 11,104,945.
Application 16/290,736 is a continuation of application No. 15/396,132, filed on Dec. 30, 2016, granted, now 10,221,448, issued on Mar. 5, 2019.
Application 15/396,132 is a continuation in part of application No. 15/057,343, filed on Mar. 1, 2016, granted, now 10,011,869, issued on Jul. 3, 2018.
Claims priority of provisional application 62/129,360, filed on Mar. 6, 2015.
Prior Publication US 2022/0002784 A1, Jan. 6, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 19/34 (2006.01); C12Q 1/686 (2018.01)
CPC C12Q 1/686 (2013.01) 17 Claims
 
1. 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 second nucleic acid sequence further comprises a full sequence of the second forward primer (F2), in between the second tag (t1) and the first reverse primer (R1).