| CPC C12P 19/34 (2013.01) [C12N 15/115 (2013.01); C12N 2310/16 (2013.01)] | 19 Claims |
|
1. A method comprising:
a) contacting at least one first plurality of solid supports and at least one first plurality of anchor primers, under conditions that allow for the attachment of a 3′ terminus of at least one anchor primer of the at least one first plurality of anchor primers to at least one solid support of the at least one first plurality of solid supports to produce at least one first anchor primer-substrate complex, wherein the anchor primers in the first plurality of anchor primers comprise at least one deoxyuridine at the 5′ terminus;
b) contacting the at least one first anchor primer-substrate complex and at least one first plurality of N-mers under conditions that append a 3′ terminus of at least one N-mer of the at least one first plurality of N-mers to a 5′ terminus of the at least one first anchor primer-substrate to produce at least one first extended anchor primer-substrate complex;
c) contacting the at least one first extended anchor primer-substrate complex and at least one second plurality of N-mers under conditions that append a 3′ terminus of at least one N-Mer of the at least one second plurality of N-mers to a 5′ terminus of the at least one first extended anchor primer-substrate complex to produce at least one first donor complex;
d) contacting at least one second plurality of solid supports and at least one second plurality of anchor primers, under conditions that allow for the attachment of a 3′ terminus of at least one anchor primer of the at least one second plurality of anchor primers to at least one solid support of the at least one second plurality of solid supports to produce at least one second anchor primer-substrate complex;
e) contacting the at least one second anchor primer-substrate complex and at least one third plurality of N-mers under conditions that append a 3′ terminus of at least one N-mer of the at least one third plurality of N-mers to a 5′ terminus of the at least one second anchor primer-substrate to produce at least one second extended anchor primer-substrate complex;
f) contacting the at least one second extended anchor primer-substrate complex and at least one fourth plurality of N-mers under conditions that append a 3′ terminus of at least one N-mer of the at least one fourth plurality of N-mers to a 5′ terminus of the at least one second extended anchor primer-substrate complex to produce at least one first target complex;
g) releasing at least one composition comprising the at least one N-mer of the at least one first plurality of N-mers and the at least one N-mer of the at least one second plurality of N-mers from the at least one first donor complex to produce at least one released intermediate complex by contacting the at least one first donor complex with a combination of uracil DNA glycosylase and at least one of DNA glycosylate-lyase, endonuclease VIII, Endo IV and APE-1; and
h) contacting the at least one first target complex and the at least one released intermediate complex under conditions that append a 3′ terminus of the at least one released intermediate complex to a 5′ terminus of the at least one target complex to produce at least one first extended target complex,
wherein the anchor primers of the first and second pluralities of anchor primers comprise deoxyribose 5′ (DNA) terminus, and
wherein the N-mers of the first, second, third and fourth pluralities of N-mers are chimeric N-mers comprising a deoxyribose 5′ (DNA) terminus and a ribose 3′ (RNA) terminus.
|