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Method for attaching a counter sequence to a nucleic acid sample

  • US 9,670,529 B2
  • Filed: 02/26/2013
  • Issued: 06/06/2017
  • Est. Priority Date: 02/28/2012
  • Status: Active Grant
First Claim
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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 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.

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