Patent ID: 9518293
Filing Date: 2016-12-13
CPC Classification: C12Q,G16B

Claim Text:
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 cells; d. 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; and 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.