Methods for the identification and characterization of double-strand break sites and compositions and uses thereof
AssigneesPioneer Hi-Bred International, Inc., Vilnius University
InventorsStephane Deschamps, Virginijus Siksnys, Joshua K Young, Mindaugas Zaremba
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Abstract
Methods and compositions are provided for the identification, detection, characterization, and/or utilization of double strand breaks in a target polynucleotide; the identification, detection, characterization, and/or utilization of cutting sites for double-strand-break-inducing agents; and the identification, detection, characterization, and/or utilization of double-strand-break-inducing agents.
Claims64
A method for characterizing a double-strand-break-inducing agent cleavage site of an isolated, purified polynucleotide, comprising:
- (a) adding phosphatase to the isolated, purified polynucleotide,
- (b) contacting the phosphatase-treated polynucleotide from (a) with a double-strand-break-inducing agent to create a library of polynucleotides,
- (c) optionally adding an adenine to the 3′ ends of the polynucleotides of the library, and
- (d) ligating an adapter to the polynucleotides of the library, wherein the adapter comprises a nucleotide that is complementary to a terminal unpaired nucleotide of the polynucleotides of (b) or (c);
- further comprising sequencing said library of polynucleotides, identifying at least one double-strand-break site, and assessing at least one qualitative characteristic or quantitative characteristic;
- wherein the phosphatase addition results in an increased number of double-strand-break sequence reads compared to a control method not comprising the addition of phosphatase.
A method for characterizing a double-strand-break-inducing agent cleavage site, comprising:
- (a) adding phosphatase to a sample of isolated, purified polynucleotide,
- (b) contacting the phosphatase-treated polynucleotide from (a) with the double-strand-break-inducing agent to create a first library of polynucleotides,
- (c) ligating a double-stranded first adapter to the polynucleotides of the first library to create a second library of polynucleotides, wherein one strand of said double-stranded first adapter comprises a molecule for the purification of the polynucleotide,
- (d) removing the nicks and reconstituting a restriction endonuclease site to create a third library of polynucleotides,
- (e) shearing the polynucleotides of the third library to create a fourth library of fragmented polynucleotides,
- (f) capturing the fragmented polynucleotides of the fourth library that comprise the ligated first adapter,
- (g) cleaving the captured fragmented polynucleotides with a restriction endonuclease capable of recognizing the site introduced in (d) to create a fifth library of polynucleotides,
- (h) Adding lambda exonuclease to the polynucleotides of the fifth library and to create a sixth library of single stranded polynucleotides,
- (i) synthesizing complementary strands to the single stranded polynucleotides of the sixth library to create a seventh library,
- (j) purifying, amplifying, and size-selecting the polynucleotides of the seventh library,
- (k) sequencing the polynucleotides of the seventh library,
- (l) identifying at least one double-strand-break site, and
- (m) assessing at least one qualitative or quantitative characteristic of said double-strand-break-inducing site;
- wherein the phosphatase addition results in an increased number of double-strand-break sequence reads compared to a control method not comprising the addition of phosphatase.
A method for characterizing a double-strand-break-inducing agent cleavage site of an isolated, purified polynucleotide, comprising:
- (a) adding phosphatase to the isolated, purified polynucleotide, wherein the isolated, purified polynucleotide is of high molecular weight,
- (b) contacting the phosphatase-treated polynucleotide from (a) with a double-strand-break-inducing agent to create a library of polynucleotides,
- (c) adding an adenine to the 3′ ends of the polynucleotides of the library,
- (d) ligating an adapter to the polynucleotides of the library, wherein the adapter comprises a nucleotide that is complementary to a terminal unpaired nucleotide of the polynucleotides of (b) or (c), and
- (e) sequencing said library of polynucleotides and identifying at least one double-strand-break site, and
- (f) assessing at least one qualitative characteristic or quantitative characteristic of the double-strand-break site;
- wherein the phosphatase addition results in an increased number of double-strand-break sequence reads compared to a control method not comprising the addition of phosphatase.
Dependencies are inferred from the claim text.