Methods relating to the detection of recurrent and non-specific double strand breaks in the genome

• US 10,640,820 B2
• Filed: 11/20/2015
• Issued: 05/05/2020
• Est. Priority Date: 11/20/2014
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation
• Original Assignee: Children's Medical Center Corporation
• Inventors: Alt, Frederick W., Frock, Richard L., Hu, Jiazhi, Meyers, Robin M.
• Primary Examiner(s): Mummert, Stephanie K
• Application Number:
  US15/527,790
• Publication Number:
  US 20180346977A1
• Time in Patent Office: Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/303 Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

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.

Claims

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.

2. The method of claim 1, wherein the first locus-specific primer comprises an affinity tag.

3. The method of claim 2, wherein the method further comprises isolating the PCR products produced in step (e) by affinity purification.

4. The method of claim 1, wherein the affinity tag is biotin and affinity purification comprises binding biotin with streptavidin.

5. The method of claim 1, wherein the affinity purification comprises binding the PCR products produced in step (d) to a substrate.

6. The method of claim 1, wherein the primers used for the nested PCR step comprise barcode sequences.

7. The method of claim 1, wherein the fragmenting is performed by sonication;

restriction digest;

randomly shearing genomic DNA; and

/or with a frequently cutting restriction enzyme.

8. The method of claim 1, wherein ligating the single-stranded PCR products to an adapter comprises contacting the PCR product with a population of adapters having the same distal portion and random proximal portion sequences.

9. The method of claim 1, wherein the adaptor comprises barcode sequences between distal and proximal portions.

10. The method of claim 1, wherein the PCR products produced in step (i) are size selected prior to sequencing.

11. The method of claim 1 wherein the agent is selected from the group consisting of:

Cas9 nuclease;

Cas9;

gRNA nuclease;

a nuclease;

a custom nuclease;

a meganuclease;

a TALEN;

a zinc-finger nuclease;

a CRISPR;

a Cpf1 CRISPR effector;

an integrating virus or viral vector;

an endonuclease;

a chemotherapeutic; and

radiation.

12. The method of claim 1, wherein the agent will generate at least one DSB within 400 bp of the hybridization target of the locus-specific primer.

13. The method of claim 1, wherein the agent will generate at least one DSB on the same chromosome as the hybridization target of the locus-specific primer.

14. The method of claim 1, wherein the cell is exposed to two agents, wherein a first agent will generate at least one DSB on the same chromosome as a DSB generated by a second agent.

15. The method of claim 1, further comprising a step of inserting into a cell to be analyzed at least one target sequence for the agent that is known to be absent in the genome of the cell to be analyzed prior to step (a) of claim 1.

16. The method of claim 1, wherein the cells are allowed to divide for at least 12 hours.

17. The method of claim 1, wherein the cell is a mammalian or plant cell.

18. The method of claim 1, wherein the cell division step (b) or the blocking digestion step (h) is omitted.

19. The method of claim 1, wherein end repair is not performed between steps (d) and (e).

20. The method of claim 1, wherein the fragmenting is performed by sonication.