Patent ID: 8512956

Claim:
A method comprising: (a) denaturing a double-stranded target DNA; (b) annealing to the target DNA and extending with a DNA polymerase comprising strand displacement activity, a first primer comprising a DNA segment and a 5′ RNA segment, wherein a 3′ portion of the primer comprises a random sequence, and a 5′ portion of the primer comprises a sequence (A), which is not complementary to the target DNA; to form a first primer extension product hybridized to the target DNA and comprising the sequence (A) at its 5′ end; (c) separating the first primer extension product from the target DNA; (d) annealing to the first primer extension product and extending a second primer comprising a 3′ complementary DNA region that comprises a random sequence, wherein the second primer is a tailed primer comprising a 5′ sequence (B), to form a double-stranded product comprising the first primer extension product and a second primer extension product, whereby a double-stranded product with a DNA/RNA heteroduplex at one end is generated; (e) cleaving the RNA in the heteroduplex from the first primer extension product such that a portion of the second primer extension product that is complementary to the sequence (A), a sequence (A′), is single stranded; (f) annealing to the second primer extension product an oligonucleotide comprising a 3′-DNA segment that is complementary to the sequence (A′) and a 5′ RNA segment comprising a sequence (C); (g) extending the oligonucleotide along the second primer extension product to form an oligonucleotide extension product comprising a sequence (B′), complementary to the sequence (B) of the second primer extension product wherein a double-stranded DNA product is formed; (h) denaturing the double-stranded DNA product of step (g); (i) attaching the oligonucleotide extension product to a solid support by annealing the sequence (B′) to a bead or isolated area comprising the sequence (B); and (j) extending the sequence (B) on the solid support to produce a third primer extension product hybridized to the oligonucleotide extension product, comprising a 3′ sequence (A′) and (C′), whereby a DNA/RNA heteroduplex at one end is generated.