US 11,884,971 B2
Compositions and techniques for nucleic acid primer extension
Denis Malyshev, La Jolla, CA (US); and Arnold Oliphant, Morgan Hill, CA (US)
Assigned to Pacific Biosciences of California, Inc., Menlo Park, CA (US)
Filed by Pacific Biosciences of California, Inc., Menlo Park, CA (US)
Filed on Aug. 2, 2021, as Appl. No. 17/391,423.
Application 17/391,423 is a continuation of application No. 16/265,942, filed on Feb. 1, 2019, abandoned.
Claims priority of provisional application 62/626,836, filed on Feb. 6, 2018.
Prior Publication US 2022/0136045 A1, May 5, 2022
Int. Cl. C12Q 1/6853 (2018.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6853 (2013.01) [C12Q 1/6869 (2013.01)] 19 Claims
 
1. A method of characterizing a nucleic acid, comprising
(a) extending a primer-template nucleic acid hybrid comprising a primer and a template nucleic acid with extendable nucleotide cognates and a nucleotide cognate comprising a reversible terminator moiety to produce an extended primer-template hybrid comprising the reversible terminator moiety;
(b) forming a stabilized ternary complex comprising the extended primer-template hybrid, a polymerase and a nucleotide cognate of the next base in the template nucleic acid;
(c) detecting the stabilized ternary complex to distinguish the next base;
(d) deblocking the extended primer-template hybrid;
(e) repeating steps (a) through (d) using the extended primer-template hybrid as the primer-template nucleic acid hybrid to determine a series of base multiplets in the template nucleic acid;
(f) repeating steps (a) through (e) to determine at least 3 different series of base multiplets in the template nucleic acid each time; and
(g) combining at least four different series of base multiplets to determine a sequence of the nucleic acid.