×

Methods and compositions for sequencing modified nucleic acids

  • US 9,238,836 B2
  • Filed: 03/15/2013
  • Issued: 01/19/2016
  • Est. Priority Date: 03/30/2012
  • Status: Active Grant
First Claim
Patent Images

1. A method for identifying sequence differences between two nucleic acids from different sources, the method comprising:

  • a) providing a first nucleic acid from a first sample;

    b) providing a second nucleic acid from a second sample;

    c) treating the first nucleic acid to produce a modified nucleic acid;

    d) denaturing the modified nucleic acid and the second nucleic acid;

    e) annealing the modified nucleic acid to the second nucleic acid, thereby producing hybrid nucleic acids that comprise a modified strand from the first sample and an unmodified strand from the second sample, and further wherein a subset of the hybrid nucleic acids comprise non-complementary regions of one or more base pairs due to sequence differences between the modified strand and the unmodified strand;

    f) binding a non-complementary-region-specific binding agent selected from the group consisting of a single-strand-specific binding agent and a mismatch-repair agent to the subset of the hybrid nucleic acids, wherein the non-complementary-region-specific binding agent comprises a selectable tag;

    g) capturing the subset of the hybrid nucleic acids bound to the non-complementary-region-specific binding agent using the selectable tag;

    h) removing nucleic acids not bound to the non-complementary-region-specific binding agent;

    i) subjecting the subset of the hybrid nucleic acids to a single type of sequencing reaction in which both the first and second strands of the hybrid nucleic acids are sequenced and both sequence data and modification data are provided in a single sequence read;

    j) for each sequence read, analyzing the sequencing data to identify said non-complementary regions; and

    k) for each sequence read, analyzing the modification data to determine which subset of said sequence read corresponds to the modified strand and which subset corresponds to the unmodified strand, thereby identifying sequence differences between the first nucleic acid from the first sample and the second nucleic acid from the second sample.

View all claims
  • 1 Assignment
Timeline View
Assignment View
    ×
    ×