US 11,946,099 B2
Elimination of primer-primer interactions during primer extension
Brian Godwin, Livermore, CA (US)
Assigned to ROCHE SEQUENCING SOLUTIONS, INC., Pleasanton, CA (US)
Filed by Roche Sequencing Solutions, Inc., Pleasanton, CA (US)
Filed on Feb. 23, 2021, as Appl. No. 17/183,255.
Application 17/183,255 is a continuation of application No. 16/080,151, granted, now 10,961,567, previously published as PCT/EP2017/053922, filed on Feb. 21, 2017.
Claims priority of provisional application 62/299,988, filed on Feb. 25, 2016.
Prior Publication US 2021/0254146 A1, Aug. 19, 2021
Int. Cl. C12Q 1/6848 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6809 (2018.01); C12Q 1/6811 (2018.01); C12Q 1/6855 (2018.01); C12Q 1/686 (2018.01)
CPC C12Q 1/6848 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6809 (2013.01); C12Q 1/6811 (2013.01); C12Q 1/6855 (2013.01); C12Q 1/686 (2013.01); C12Q 2525/101 (2013.01); C12Q 2525/186 (2013.01)] 10 Claims
 
1. A method of amplifying a target nucleic acid in a sample with reduced primer-primer interaction comprising:
(a) a primer extension step, wherein the sample is contacted with: (i) a nucleic acid polymerase, and (ii) a first primer which is a single-stranded oligonucleotide comprising at least one modified nucleotide that stalls nucleotide incorporation by the polymerase, the first primer comprising two arms complementary to a target sequence separated by a linker sequence non-complementary to the target sequence, such that a gap is created between the ends of the two arms when the first primer hybridizes to the target nucleic acid, wherein the polymerase fills the gap between the ends of the two arms; and
(b) an exponential amplification step wherein the sample is contacted with a second primer complementary to the primer extension product and a polymerase tolerant of the modified nucleotide.