US 12,134,766 B2
Methods for generating circular nucleic acid molecules
Matthew Kellinger, San Diego, CA (US); Sinan Arslan, San Diego, CA (US); Michael Previte, San Diego, CA (US); and Junhua Zhao, San Diego, CA (US)
Assigned to ELEMENT BIOSCIENCES, INC., San Diego, CA (US)
Filed by Element Biosciences, Inc., San Diego, CA (US)
Filed on Jan. 11, 2023, as Appl. No. 18/153,268.
Application 18/153,268 is a continuation of application No. 17/320,042, filed on May 13, 2021.
Application 17/320,042 is a continuation of application No. PCT/US2019/061871, filed on Nov. 15, 2019.
Claims priority of provisional application 62/767,943, filed on Nov. 15, 2018.
Prior Publication US 2023/0167434 A1, Jun. 1, 2023
Int. Cl. C12N 15/10 (2006.01); C12N 9/12 (2006.01); C12Q 1/6869 (2018.01)
CPC C12N 15/1068 (2013.01) [C12N 9/12 (2013.01); C12N 9/1241 (2013.01); C12Q 1/6869 (2013.01)] 20 Claims
 
1. A method for determining a sequence, comprising:
(a) providing a concatemer, wherein said concatemer comprises (i) an adapter sequence configured to couple to a single surface-bound oligonucleotide and (ii) a strand comprising two or more repeats of an identical sequence, and wherein said two or more repeats of said identical sequence comprise a target sequence and an enzyme recognition site;
(b) coupling said concatemer to a surface;
(c) hybridizing a primer sequence complementary to said target sequence, thereby producing a primed nucleic acid sequence;
(d) extending said primed nucleic acid sequence;
(e) digesting said strand;
(f) hybridizing another primer sequence to another target sequence in another strand of said concatemer, wherein said another strand is complementary to said strand, thereby producing another primed nucleic acid sequence, and wherein said strand and said another strand are formed by rolling circle amplification (RCA) and multiple displacement amplification (MDA); and
(g) extending said another primed nucleic acid sequence.