HIGH THROUGHPUT GENOME-WIDE TRANSLOCATION SEQUENCING
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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Abstract
Provided are methods for high-throughput screening to determine locations of double-stranded DNA breaks (DSBs) and translocations in genomes caused by different agents, such as enzymes.
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Citations
49 Claims
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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:
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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; 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. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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2-25. -25. (canceled)
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37. A method for high throughput, genome-wide translocation sequencing (HTGTS) and identification of double-stranded DNA break (DSB) locations comprising the steps of:
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a. exposing a cell to an agent known or suspected to be capable of producing a 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 sample by ligating the fragmented DNA at a concentration favoring intra-molecular ligation; f. digesting the ligated DNA sample with a blocking enzyme; g. producing nested PCR products by performing a nested PCR with locus-specific primers; h. sequencing the nested PCR products; i. aligning the sequences against a reference sequence to identify chromosomal locations of the translocations and DSBs. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49)
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Specification