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US 2014/0234847 A1Published application
High throughput genome-wide translocation sequencing
AssigneeChildren's Medical Center Corporation
InventorsFrederick W. Alt, Yu Zhang, Roberto Chiarle, Monica Gostissa
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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.
Claims26
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;
- 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.
6 more elements
2-25. (canceled)
A method for high throughput, genome-wide translocation sequencing (HTGTS) and identification of double-stranded DNA break (DSB) locations comprising the steps of:
- 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.
6 more elements
Dependencies are inferred from the claim text.