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HIGH THROUGHPUT GENOME-WIDE TRANSLOCATION SEQUENCING

  • US 20140234847A1
  • Filed: 07/06/2012
  • Published: 08/21/2014
  • Est. Priority Date: 07/07/2011
  • 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 12 hours;

    c. extracting genomic DNA from the cellsd. producing a fragmented DNA sample by fragmenting the DNA of the cell with a frequently cutting restriction enzyme;

    e. producing a ligated DNA product by ligating an asymmetric adapter to the fragmented DNA sample, wherein the asymmetric adapter comprises a sequence that is designed to anneal to the DNA end generated by the frequently cutting restriction enzyme and contains a stretch of known DNA sequence that can be used to design a PCR primer for a nested PCR amplification;

    f. digesting the ligated DNA products with an enzyme to block amplification of germline or unrearranged targeted alleles;

    g. producing nested PCR products by performing nested-PCR with adapter- and locus-specific primers using the digested ligated DNA product thereby amplifying the nucleic acid sequences surrounding the junctions around the DSBs;

    h. producing sequenced nested PCR products by sequencing the nested PCR products;

    i. aligning the sequenced nested PCR products against a reference sequence to identify chromosomal locations of the translocations and the chromosomal locations of the DSBs.

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