Method for the isolation of double-strand breaks
View Patent ↗The invention relates to a method for determining the number and nature of DNA double-strand breaks (DSBs) in a nucleic acid sample, ideally gDNA; a kit of parts for performing the method including at least a plurality of oligonucleotides for ligating to the nucleic acid sample and providing at least a first hybridization site (RD1 SP or RD2 SP) to which at least one read sequencing primer can hybridise; and oligonucleotides for use in the kit and method.
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.
2. The method of claim 1 , wherein the further sequence comprises a sequence for enabling bridge amplification.
3. The method of claim 1 , wherein the first oligonucleotide of the second pair of oligonucleotides does not comprise a binding sequence for separating said fragmented nucleic acid.
4. The method of claim 1 , wherein the method is performed in the order: step ii), step iii), step i), step iv), step v), and step vi), wherein after fragmenting in step ii), the nucleic acid is first exposed to the second pair of oligonucleotides in step iii) and the nucleic acid is then exposed to the first pair of oligonucleotides in step i).
5. The method of claim 1 , wherein said nucleic acid sample comprises genomic DNA (gDNA).
6. The method of claim 1 , wherein
the 5′ binding feature of the first oligonucleotide of the first pair of oligonucleotides,
the 3′ binding feature of the second oligonucleotide of the first pair of oligonucleotides,
the 5′ binding feature of the first oligonucleotide of the second pair of oligonucleotides, and/or
the 3′ binding feature of the second oligonucleotide of the second pair of oligonucleotides
comprises a phosphate group, a triphosphate ‘T-tail’, a triphosphate ‘A-tail’, at least one random N nucleotide, or a plurality of N nucleotides.
7. The method of claim 6 , wherein the triphosphate ‘T-tail’ is a deoxythymidine triphosphate ‘T-tail’.
8. The method of claim 6 , wherein the triphosphate ‘A-tail’ is a deoxyadenosine triphosphate ‘A-tail’.
9. The method of claim 1 , wherein
the 3′ and/or 5′ protective feature of the first oligonucleotide of the first pair of oligonucleotides,
the 3′ and/or 5′ protective feature of the second oligonucleotide of the first pair of oligonucleotides,
the 3′ and/or 5′ protective feature of the first oligonucleotide of the second pair of oligonucleotides, and/or
the 3′ and/or 5′ protective feature of the second oligonucleotide of the second pair of oligonucleotides
comprises a feature that provides resistance to any one or more of the following: phosphorylation activity, phosphatase activity, terminal transferase activity, nucleic acid hybridization, endonuclease activity, exonuclease activity, ligase activity, polymerase activity, and protein binding.
10. The method of claim 1 wherein
the 3′ and/or 5′ protective feature of the first oligonucleotide of the first pair of oligonucleotides,
the 3′ and/or 5′ protective feature of the second oligonucleotide of the first pair of oligonucleotides,
the 3′ and/or 5′ protective feature of the first oligonucleotide of the second pair of oligonucleotides, and/or
the 3′ and/or 5′ protective feature of the second oligonucleotide of the second pair of oligonucleotides
comprises a phosphorothioate linkage, a dideoxynucleotide or a covalent block, a phosphoramidite, or a C3 Spacer phosphoramidite (3SpC3).
11. The method of claim 1 , wherein said first and second oligonucleotides of the first pair of oligonucleotides and the first and second oligonucleotides of the second pair of oligonucleotides comprise an index feature that is a particular sequence of nucleotides that enables the origin of pooled samples to be determined.
12. The method of claim 1 , wherein:
said first oligonucleotide of the first pair of oligonucleotides, reading 5′ to 3′, comprises the 5′ binding feature, the first hybridization site to which a sequencing primer can bind, the binding sequence for separating said DSB from a pool of DSBs, and a 3′ protective feature; and/or
said second oligonucleotide of the second pair of oligonucleotides, reading 5′ to 3′ comprises the sequence for enabling bridge amplification, the second hybridization site to which a sequencing primer can bind, and a 3′ binding feature.
13. The method of claim 1 , wherein the first oligonucleotide of the first pair of oligonucleotides comprises a 3′ protective feature; and wherein the first oligonucleotide of the second pair of oligonucleotides comprises a 3′ protective feature.
14. The method of claim 1 , wherein:
said first pair of oligonucleotides comprises the first oligonucleotide having SEQ ID NO. 1 and the second oligonucleotide having SEQ ID NO. 2; or an oligonucleotide that shares at least 80% identity or homology with SEQ ID NO. 1 or 2; and/or
said second pair of oligonucleotides comprises the first oligonucleotide having SEQ ID NO. 3 and the second oligonucleotide having SEQ ID NO. 4; or an oligonucleotide that shares at least 80% identity or homology with SEQ ID NO. 3 or 4.
15. The method of claim 1 , wherein said second oligonucleotide of said second pair of oligonucleotides comprises any one of the following sequences: SEQ ID NOs. 4-28; or an oligonucleotide that shares at least 80% identity or homology with one of SEQ ID NOs. 4-28.
16. The method of claim 1 , wherein said sample is mammalian or human.
17. The method of claim 1 , wherein said ligation in part i) occurs in situ or in vitro using a cell or tissue sample.
18. The method of claim 1 , wherein said sample is exposed to a permeabilizing agent and/or at least one agent for performing adenosine tail repair before step i) is undertaken.
19. The method of claim 1 , wherein part i) further comprises extracting genomic DNA (gDNA) from said sample prior to performing the subsequent steps.
20. The method of claim 1 , wherein said method further comprises after part ii) and/or part iv), removing fragments whose size is less than about 100 bp, or less than about 150 bp, and/or retaining fragments whose size is greater than about 150 bp.
21. The method of claim 1 , wherein part vi) comprises bridge amplification where the single strands separated under part v) are clonally amplified on a substrate that has anchored thereon oligonucleotides/binding sites for the binding sequence of the first oligonucleotide of the first pair of oligonucleotides and the further sequence of the second oligonucleotide of the second pair of oligonucleotides.
22. The method of claim 1 , wherein prior to performing the claimed method, a sample containing or suspected of containing a single strand break is ligated or broken to ensure the single strand break is converted into a double strand break.