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Methods relating to the detection of recurrent and non-specific double strand breaks in the genome

  • US 10,640,820 B2
  • Filed: 11/20/2015
  • Issued: 05/05/2020
  • Est. Priority Date: 11/20/2014
  • Status: Active Grant
First Claim
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1. A method for high throughput, genome-wide translocation sequencing (HTGTS) and detection of double-stranded DNA break (DSB) locations, the method comprising the steps of:

  • a. exposing a cell to an agent known or suspected of being capable of producing at least one DSB;

    b. optionally, allowing the cell to divide for at least a half cell cycle after exposure;

    c. extracting genomic DNA from the cells;

    d. optionally, producing a fragmented DNA sample;

    e. producing a single-stranded PCR product by Linear Amplification Mediated (LAM)-PCR with a first locus-specific primer;

    f. producing a ligated DNA product by ligating the single-stranded PCR product produced in step (e) to an adapter, wherein the adapter comprises;

    a distal portion of known DNA sequence that can be used to design PCR primers for a nested PCR amplification;

    a proximal portion of random nucleotides; and

    a 3′

    overhang;

    g. producing a nested PCR product by performing a nested-PCR with an adapter- and a locus-specific primer using the ligated DNA product thereby amplifying the nucleic acid sequence surrounding the junction around the at least one DSB;

    h. optionally, digesting the ligated DNA sample with a blocking enzyme;

    i. producing a sequenced nested PCR product by sequencing the nested PCR product;

    j. aligning the sequenced nested PCR product against a reference sequence to identify a chromosomal location of the translocation and the chromosomal location of the at least one DSB.

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