Method for Attaching a Counter Sequence to a Nucleic Acid Sample

摘要

Described herein is a method for adding a counter sequence to the individual polynucleotide molecules of an initial nucleic acid sample. After addition of the counter sequence, the sample may be amplified and the number of initial target molecules in the sample can be estimated by counting the number of counter sequences associated with the amplified target molecules.

主权项

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:
(i) region A provides a primer binding site when copied and is the same in every primer of the population of forward primers;(ii) region Y varies between the different primers of the primer population and provides a counter sequence; and(iii) region Z is complementary to a first site in a target polynucleotide in a population of template nucleic acid molecules and is the same in every primer of said population of primers; and said hybridizing is done in the presence of a second primer comprising the sequence of region A and said 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 target polynucleotide 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.