Compositions and methods for detecting nucleic acid regions

Patent metadata

Field	Value
Publication number	US-10676734-B2
Application number	US-201715646851-A
Country	US
Kind code	B2
Filing date	Jul 11, 2017
Priority date	Jul 12, 2016
Publication date	Jun 9, 2020
Grant date	Jun 9, 2020

Abstract

Provided are methods, compositions, reagents, kits that are useful for detecting a specific nucleic acid region in genome with high efficiency and high sensitivity.

First claim

What is claimed is:

1. A method for detecting off-target insertion of a donor nucleic acid molecule into the genome of a cell, the method comprising:
(i) contacting the cell with (a) a gene editing reagent and (b) the donor nucleic acid molecule under conditions that allow for generation of a double-stranded break at a predetermined genetic locus in the genome of the cell;
(ii) collecting nucleic acid from the cell of (i) to generate a nucleic acid sample;
(iii) fragmenting the nucleic acid present in the nucleic acid sample to generate a plurality of nucleic acid fragments;
(iv) ligating a first nucleic acid adapter to termini of the plurality of nucleic acid fragments, thereby forming a plurality of adapted nucleic acid fragments;
(v) amplifying the plurality of adapted nucleic acid fragments by a first polymerase chain reaction procedure using a plurality of primers, wherein the plurality of primers comprise (a) a first primer that is capable of hybridizing to at least one of the nucleic acid adapters and (b) a second primer that is capable of hybridizing to the donor nucleic acid molecule and wherein the second primer contains a 5′ terminal phosphate, thereby forming a plurality of amplified 5′-phosphate nucleic acid segments;
(vi) ligating a second nucleic acid adapter to the plurality of amplified 5′-phosphate nucleic acid segments, wherein the second nucleic acid adapter is ligated to the plurality of amplified 5′-phosphate nucleic acid segments, thereby forming a plurality of adapter nucleic acid segments;
(vii) amplifying the plurality of adapter nucleic acid segments by a second polymerase chain reaction procedure, thereby forming a plurality of amplified adapter nucleic acid segments;
(viii) attaching a separation agent to the plurality of amplified adapter nucleic acid segments, thereby forming a plurality of separation agent adapter nucleic acid segments;
(ix) separating the separation agent adapter nucleic acid segments from nucleic acid molecules that are not attached to the separation agent, thereby generating a population of separation agent adapter nucleic acid segments; and
(x) sequencing individual members of the population of separation agent adapter nucleic acid segments, thereby detecting off-target insertion of the donor nucleic acid molecule into the genome of the cell.

2. The method of claim 1 , wherein the gene editing reagent comprises one or more zinc finger-FokI fusion protein, one or more transcription activator-like effector nuclease (TALEN), or one or more Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleic acid complex.

3. The method of claim 1 , wherein the plurality of nucleic acid fragments comprise chromosomal nucleic acid.

4. The method of claim 1 , wherein the plurality of nucleic acid fragments are about 100 bp to about 1000 bp in length.

5. The method of claim 1 , wherein the plurality of nucleic acid fragments are blunt-ended.

6. The method of claim 1 , wherein the cell is a mammalian cell.

7. The method of claim 6 , wherein the mammalian cell is a human cell.

8. The method of claim 1 , wherein the fragmenting of the nucleic acid is performed by sonication or by contacting with a DNAse.

9. The method of claim 1 , wherein the second nucleic acid adapter is a barcode adapter.

10. The method of claim 1 , wherein the plurality of amplified adapter nucleic acid segments comprises a single strand tail on at least one end.

11. The method of claim 10 , wherein the separation agent comprises a biotinylated tag oligonucleotide that is complementary to the single strand tail.

12. The method of claim 1 , wherein the separating of the separation agent adapter nucleic acid segments from nucleic acid molecules that are not attached to the separation agent is mediated by the association of the separation agent adapter nucleic acid segments with a support.

13. The method of claim 12 , wherein the support comprises a magnetic bead.

14. The method of claim 1 , wherein the sequencing is next generation sequencing.

Assignees

• Life Technologies Corp

Inventors

• Tang Pei-Zhong

Classifications

• C12Q1/6855

  Ligating adaptors · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q2521/501

  Ligase · CPC title

• C12Q1/6853 Primary

  using modified primers or templates · CPC title