Patent ID: 9670529
Date: 2017-06-06
CPC Classifications: C12Q,G16B

Claim:
1. A method of processing a nucleic acid sample, comprising, (a) hybridizing a population of forward primers of the formula 5′-A-Y-Z to a population of template nucleic acid molecules, wherein: said hybridizing is done in the presence of a second primer comprising the sequence of region A and a reverse primer and, if said reverse primer contains a 5′ tail, an optional third primer that has the sequence of said 5′ tail; (b) extending the forward primers that are hybridized to said population of template nucleic acid molecules in step (a) using said target polynucleotide as a template to produce a population of first extension products that comprise a binding site for said reverse primer; (c) hybridizing said reverse primer to the binding site for said reverse primer in the population of first extension products produced by step (b); (d) extending said reverse primer to produce a population of second extension products that comprises the complement of region A and the complement of region Y, wherein the complement of region Y is different in each molecule in said population of second extension products; (e) selectively disabling, after step (d) any forward primers that are not extended; and (f) amplifying said population of second extension products by PCR using said second primer comprising region A and (i) said reverse primer or (ii) said third primer, to produce a population of PCR products in which clonally-related products are tagged with the same counter sequence and products that are not clonally related are tagged with a different sequence relative to one another, wherein said steps (a) to (f) are done in a closed vessel and no additional reagents are added to the vessel during the method.