| CPC C12Q 1/6883 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6874 (2013.01); C12Q 2600/156 (2013.01)] | 22 Claims |
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1. A method for identifying DNA double-strand breaks (DSBs) in a sample comprising a nucleic acid, comprising
i) exposing the sample of nucleic acid suspected of containing DSBs, under ligation conditions, to a first pair of oligonucleotides, wherein a first oligonucleotide of the first pair of oligonucleotides comprises a 5′ binding feature that enables ligation of said first oligonucleotide to a first strand of a DSB, a first hybridization site to which a first sequencing primer can bind, and a binding sequence for separating said DSB from a pool of DSBs; and a second oligonucleotide of the first pair of oligonucleotides that is complementary to said first oligonucleotide of the first pair and comprises a 3′ binding feature that enables ligation of said second oligonucleotide to a second strand of said DSB; wherein either or both of said oligonucleotides comprise, a 3′ and/or 5′ protective feature;
ii) fragmenting the nucleic acid of said sample into fragments;
iii) exposing said fragments, under ligation conditions, to a second pair of oligonucleotides, wherein a first oligonucleotide of the second pair of oligonucleotides comprises a 5′ binding feature, that enables ligation of said first oligonucleotide to a first strand of a fragmented nucleic acid and a second hybridization site to which a second sequencing primer can bind; and a second longer oligonucleotide of the second pair of oligonucleotides that is in part complementary to said first oligonucleotide of the second pair and comprises a 3′ binding feature for binding to a second strand of said fragmented nucleic acid, a sequence complimentary to said second hybridization site, and a further sequence; and wherein either or both of said oligonucleotides comprise a 3′ and/or 5′ protective feature;
iv) denaturing the fragments to provide single strand nucleic acids;
v) separating the single strand nucleic acids of part iv) into two groups: group A strands that are associated with a DSB and have ligated at a first end the first hybridization site and the binding sequence provided by the first oligonucleotide of the first pair of oligonucleotides and at another end the second hybridization site and the further sequence provided by the second oligonucleotide of the second pair of oligonucleotides and group B strands that are not associated with a DSB and do not have ligated at a first end the hybridization site and the binding sequence provided by the first oligonucleotide of the first pair of oligonucleotides, wherein the separating comprises
hybridizing the binding sequence provided by the first oligonucleotide of the first pair of oligonucleotides to a complementary binding strand anchored to a substrate in order to retain group A strands, and
removing group B strands from the substrate; and
vi) sequencing the strands of group A using at least the first sequencing primers where each sequence is equivalent to a DSB, wherein the number and nature of base pair deletions can be determined by comparing each sequence with a genome representative of a species from which the sample was taken.
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