Primers and assays for linking regions using polymerases

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 a first intermediate region,
• a polymerase,
• 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 a first overlapping sequence,
• a forward primer F2 having a first portion complementary to a starting sequence of the region B, the forward primer F2 including a second portion having a first complementary overlapping sequence that is complementary to the first overlapping sequence,
• a forward primer F4 having a first portion complementary to a starting sequence of a region D of the first DNA molecule, the region D separated from the region A by a second intermediate region, the forward primer F4 including a second portion having the first complementary overlapping sequence,
• a reverse primer R2 having a first portion complementary to an ending sequence of the region B, the reverse primer R2 including a second portion having a second overlapping sequence,
• a reverse primer R4 having a first portion complementary to an ending sequence of the region D, the reverse primer R4 including a second portion having the second overlapping sequence, and
• a forward primer F3 having a first portion complementary to a starting sequence of a region C of the first DNA molecule, the region C separated from the region B by a third intermediate region, the forward primer F3 including a second portion having the second complementary overlapping sequence;
• amplifying, using the polymerase and the reverse primer R1, the region A to obtain a first set of amplicons;
• amplifying, using the polymerase, the forward primer F2, and the reverse primer R2, the region B to obtain a second set of amplicons;
• amplifying, using the polymerase, the forward primer F4, and the reverse primer R4, the region D to obtain a third set of amplicons;
• amplifying, using the polymerase and the forward primer F3, the region C to obtain a fourth set of amplicons;
• 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 first extended amplicons that include the region A and the region B and exclude the first intermediate region;
• extending, using the polymerase, the first set of amplicons to include the region D and the third set of amplicons to include the region A, thereby obtaining second extended amplicons that include the region A and the region D and exclude the second intermediate region;
• and
• extending, using the polymerase, the second extended amplicons to include the region C, thereby obtaining third extended amplicons that include the region A, the region D, and the region C, and
• extending, using the polymerase, the first extended amplicons to include the region C, thereby obtaining fourth extended amplicons that include the region A, the region B, and the region C.