METHODS RELATING TO THE DETECTION OF RECURRENT AND NON-SPECIFIC DOUBLE STRAND BREAKS IN THE GENOME
First Claim
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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Abstract
Described herein are methods and systems relating to high throughput, genome-wide translocation sequencing (HTGTS) and/or detection of double-stranded DNA break (DSB) locations. The methods described herein can comprise generating DSBs in a nucleic acid sequence and performing nested PCR with the primers described herein. Described herein is an enhanced HTGTS approach. The assays and methods described herein permit the measurement of various DNA double-strand break (DSB) activities either intrinsic to the biological system or from outside agents.
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Citations
43 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 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. - View Dependent Claims (2, 3, 5, 6, 8, 9, 11, 14, 15, 17, 18, 19, 20, 23, 24, 31, 33, 35)
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36. A kit comprising:
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a. an agent known to cause at least one DSB in a cell; and b. a locus-specific primer that will anneal within 400 bp of the DSB generated by the agent. - View Dependent Claims (37)
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38-41. -41. (canceled)
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42. A computer implemented method for high throughput, genome-wide translocation sequencing (HTGTS) and detection of double-stranded DNA break (DSB) locations, comprising:
- on a device having one or more processors and a memory storing one or more programs for execution by the one or more processors, the one or more programs including instructions for;
aligning a sequenced nested PCR product against a reference sequence to identify a chromosomal location of a translocation and the chromosomal location of at least one DSB.
- on a device having one or more processors and a memory storing one or more programs for execution by the one or more processors, the one or more programs including instructions for;
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43-67. -67. (canceled)
Specification