US 12,264,315 B2
Target enrichment by unidirectional dual probe primer extension
Daniel Burgess, Black Earth, WI (US); Brian Christopher Godwin, Livermore, CA (US); Alexander Lovejoy, Newark, CA (US); Bronwen Miller, Cape Town (ZA); Jo-Anne Elizabeth Penkler, Cape Town (ZA); Joseph Platzer, Oakland, CA (US); and Martin Ranik, Santa Clara, CA (US)
Assigned to Roche Sequencing Solutions, Inc., Pleasanton, CA (US); and Kapa Biosystems, Inc., Wilmington, MA (US)
Filed by Roche Sequencing Solutions, Inc., Pleasanton, CA (US); and Kapa Biosystems, Inc., Wilmington, MA (US)
Filed on Aug. 15, 2019, as Appl. No. 16/542,127.
Application 16/542,127 is a continuation in part of application No. PCT/EP2018/085727, filed on Dec. 19, 2018.
Claims priority of provisional application 62/609,013, filed on Dec. 21, 2017.
Claims priority of application No. PCT/EP2018/085727 (WO), filed on Dec. 19, 2018.
Prior Publication US 2020/0032244 A1, Jan. 30, 2020
Int. Cl. C12N 15/10 (2006.01); C12Q 1/6813 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6876 (2018.01)
CPC C12N 15/1034 (2013.01) [C12N 15/1072 (2013.01); C12Q 1/6813 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6876 (2013.01)] 9 Claims
 
1. A method for enrichment of at least one target nucleic acid in a library of nucleic acids, the method comprising the following ordered steps:
(i) hybridizing a first oligonucleotide to a strand of a target nucleic acid in a library of nucleic acids, wherein the strand of the target nucleic acid is genomic DNA, wherein each of the nucleic acids in the library of the nucleic acids has a first end comprising a first adapter and a second end comprising a second adapter, wherein the first adapter is joined to the 3′ terminus of the first end and the second adapter is joined to the 5′ terminus of the second end, wherein the first oligonucleotide comprises a first portion including a first sequence complementary to a sequence of the target nucleic acid and a second portion comprising a capture sequence;
(ii) extending the hybridized first oligonucleotide of step (i) with a first polymerase, thereby producing a first primer extension complex, wherein the first primer extension complex comprises the strand of the target nucleic acid, wherein the strand of the target nucleic acid is genomic DNA, and the extended first oligonucleotide;
(iii) capturing the first primer extension complex of step (ii) by (a) hybridizing a capture oligonucleotide to the second portion of the first oligonucleotide, where the capture oligonucleotide comprises a sequence complementary to the capture sequence and capture moiety, and (b) binding the capture moiety of the hybridized capture oligonucleotide to a solid support;
(iv) enriching the captured first primer extension complex of step (iii) relative to the library of nucleic acids;
(v) hybridizing a second oligonucleotide to the strand of the target nucleic acid of the first primer extension complex of step (iv), wherein the strand of the target nucleic acid is genomic DNA, wherein the second oligonucleotide is complementary to a sequence of the target nucleic acid and hybridizes to the same strand of the target nucleic acid of the first primer extension complex at a position 5′ relative to the first oligonucleotide;
(vi) extending the hybridized second oligonucleotide of step (v) with a second polymerase, thereby producing a second primer extension complex, wherein the second primer extension complex comprises the strand of the target nucleic acid, wherein the strand of the target nucleic acid is genomic DNA, and the extended second oligonucleotide, thereby liberating the extended first oligonucleotide captured via the hybridized capture oligonucleotide from the first primer extension complex, and releasing the second primer extension complex comprising the strand of the target nucleic acid into solution; and
(vii) amplifying the target nucleic acid strand in the second primer extension complex of step (vi) with a third polymerase, a first amplification primer, and a second amplification primer, wherein the first amplification primer is complementary to the first adapter, and wherein the second amplification primer is complementary to the second adapter, wherein the amplification produces an amplified copy of the target nucleic acid, wherein the amplified copy of the target nucleic acid comprises the entire sequence of the target nucleic acid, wherein the target nucleic acid is genomic DNA, and wherein the length of the amplified copy of the target nucleic acid is longer than the lengths of the extended first oligonucleotide and the extended second oligonucleotide.