US 11,718,850 B2
Primers and assays for linking regions using polymerases
Kwan Chee Chan, Hong Kong (CN); Wanxia Gai, Shenzhen (CN); and Yuk-Ming Dennis Lo, Hong Kong (CN)
Assigned to The Chinese University of Hong Kong, Shatin (HK)
Filed by The Chinese University of Hong Kong, Shatin (HK)
Filed on May 11, 2020, as Appl. No. 16/871,754.
Claims priority of provisional application 62/846,149, filed on May 10, 2019.
Prior Publication US 2020/0354785 A1, Nov. 12, 2020
Int. Cl. C12N 15/10 (2006.01); C12Q 1/68 (2018.01)
CPC C12N 15/1093 (2013.01) [C12N 15/1031 (2013.01); C12Q 1/68 (2013.01); C12Q 2521/101 (2013.01); C12Q 2533/101 (2013.01); C12Q 2535/125 (2013.01)] 33 Claims
OG exemplary drawing
 
1. A method for connecting separated regions of DNA molecules in a biological sample of a subject, the method comprising:
partitioning a plurality of DNA molecules from the biological sample into a plurality of compartments, wherein a first compartment of the plurality of compartments includes:
a first DNA molecule having a region A and a region B separated by an intermediate region,
a polymerase having a bias for adding a particular nucleotide at an overhang position,
a reverse primer R1 having a first portion complementary to an ending sequence of the region A, the reverse primer R1 including a second portion having an overlapping sequence, and
a forward primer F2 having a first portion complementary to a starting sequence of the region B, the forward primer F2 including a complementary overlapping sequence that is complementary to the overlapping sequence;
amplifying, using the polymerase and the reverse primer R1, the region A to obtain a first set of amplicons having the particular nucleotide at a first 3′ end and having a complementary nucleotide between sequence strings corresponding to the first portion and the second portion of the reverse primer R1, the complementary nucleotide being complementary to the particular nucleotide;
amplifying, using the polymerase and the forward primer F2, the region B to obtain a second set of amplicons having the particular nucleotide at a second 3′ end and having the complementary nucleotide on an opposite end of the overlapping sequence; and
extending, using the polymerase, the first set of amplicons to include the region B and the second set of amplicons to include the region A, thereby obtaining extended amplicons that include the region A and the region B and exclude the intermediate region.