C-probe libraries for DNA target enrichment

Patent metadata

Field	Value
Publication number	US-10150960-B2
Application number	US-201313777842-A
Country	US
Kind code	B2
Filing date	Feb 26, 2013
Priority date	Feb 26, 2013
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

Abstract

Provided herein is a method for enriching a target nucleic acid molecule. In one embodiment, the method may involve hybridizing a C-probe to a strand of a target nucleic acid to produce a complex, enzymatically removing any 3′ overhanging end from the target nucleic acid of the complex to produce a 3′ hydroxyl group at the 3′ end; extending the 3′ end of the first sequence using the oligonucleotide sequence of the C-probe as a template; enzymatically removing any 5′ overhanging end from the target nucleic acid, either before or after the extending step, to produce an 5′ phosphate group at the end of the second sequence; and ligating the 5′ phosphate group at the end of the second sequence to the 3′ hydroxyl group at the end of the first sequence to produce a circular DNA molecule that contains the target sequence and the complement of the oligonucleotide sequence.

First claim

The invention claimed is:

1. A method of processing a nucleic acid, comprising:
(a) hybridizing a C-probe to a strand of a target nucleic acid to produce a complex, wherein:
(i) said strand comprises a target sequence that is flanked by a first sequence and a second sequence, and
(ii) said C-probe comprises a first region that hybridizes to said first sequence, a second region that hybridizes to said second sequence, and an oligonucleotide sequence between said first and second regions,

(b) enzymatically removing any 3′ overhang from the target nucleic acid of said complex to produce a 3′ hydroxyl group at the 3′ end of said first sequence;
(c) extending the 3′ end of said first sequence using the oligonucleotide sequence of said C-probe as a template, wherein said extending results in a 3′ hydroxyl group that is adjacent to the 5′ end of said second sequence;
(d) enzymatically removing any 5′ overhanging from the target nucleic acid, either before or after said extending of step (c), to produce a 5′ phosphate group at the end of said second sequence; and
(e) ligating the 5′ phosphate group at the end of the second sequence to the 3′ hydroxyl group that is adjacent to the 5′ end of said second sequence to produce a circular DNA molecule that contains said target sequence and the complement of said oligonucleotide sequence.

2. The method of claim 1 , wherein said method comprises:
(f) amplifying the target sequence from said circular DNA molecule using one or more primers that either hybridize with or have the same sequence as sites in the oligonucleotide sequence.

3. The method of claim 2 , further comprising:
(g) sequencing the amplification product of (f) to provide the nucleotide sequence of at least part of the target sequence.

4. The method of claim 1 , wherein the 5′ overhanging end of the target nucleic acid is enzymatically removed before step (c).

5. The method of claim 4 , wherein the 3′ and 5′ overhanging ends of the target nucleic acid are enzymatically removed using a single-strand specific bi-directional exonuclease, optionally in the presence of a polymerase.

6. The method of claim 5 , wherein said single-strand specific bi-directional exonuclease is exonuclease VII.

7. The method of claim 4 , wherein said C-probe is designed to provide G-A mismatches in the complex, and the 3′ and 5′ overhanging ends of the target nucleic acid are enzymatically removed using glycosylase and AP endonuclease activities.

8. The method of claim 4 , wherein the 3′ and 5′ overhanging ends of the target nucleic acid are enzymatically removed using exonuclease T and T7 exonuclease.

9. The method of claim 1 , wherein the 5′ overhanging end of the target nucleic acid is enzymatically removed after step (c).

10. The method of claim 1 , wherein:
the 3′ overhanging end of the target nucleic acid is enzymatically removed by the exonuclease activity of T4 DNA polymerase;
said extending step (c) is done using the polymerase activity of T4 DNA polymerase; and
the 3′ overhanging end of the target nucleic acid is enzymatically removed by a flap endonuclease.

11. The method of claim 1 , wherein the oligonucleotide sequence comprises a molecular barcode.

12. The method of claim 1 , wherein said target nucleic acid is genomic DNA.

13. The method of claim 12 , wherein the genomic DNA is randomly sheared genomic DNA that has been produced using a chemical, physical or transposase-catalyzed fragmentation method.

14. The method of claim 1 , wherein the 3′ overhanging end of the target nucleic acid is enzymatically removed by a nicking endonuclease.

15. The method of claim 1 , wherein the 5′ overhanging end of the target nucleic acid is enzymatically removed by a nicking endonuclease.

16. The method of claim 1 , wherein said target sequence is in the range of 10 nt to 1,000 nt in length.

17. The method of claim 1 , wherein the region extended in step (c) is composed of no more than three different nucleotides.

18. The method of claim 1 , wherein the 3′ end of said C-probe is blocked to prevent the addition of nucleotides by a polymerase or a ligase.

19. The method of claim 18 , wherein the 3′ end of said C-probe comprises a dideoxy nucleotide and a phosphothioate linkage.

20. A kit for processing a target nucleic acid, comprising:
a C-probe comprising a first region that hybridizes to a first sequence in a strand of said target nucleic acid, a second region that hybridizes to a second sequence in said strand, and an oligonucleotide sequence between said first and second regions, wherein the 3′ end of said C-probe is blocked to prevent the addition of nucleotides by a polymerase or a ligase,
one or more enzymes that remove 5′ and 3′ single stranded overhangs; and
a DNA ligase.

Assignees

• Agilent Technologies Inc

Inventors

• Peter Brian Jon

Classifications

• C12N15/10 Primary

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title