Restriction Enzyme-Free Target Enrichment

• US 20150307875A1
• Filed: 10/03/2013
• Published: 10/29/2015
• Est. Priority Date: 12/06/2012
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Leproust, Emily Marine, Ong, Joseph Hoang Hai, Happe, Scott Robert, Barboza, Julia
• Granted Patent: US 10,072,260 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides

Abstract

Provided herein are various methods for enriching a target fragment that is present in randomly sheared genomic DNA. In some embodiments, the method may involve hybridizing randomly sheared genomic DNA to a halo probe to produce a first circular complex, and then enzymatically digesting the overhanging ends of the genomic fragment. Other embodiments may include hybridizing randomly sheared genomic DNA to an RNA oligonucleotide that comprises a region that hybridizes to a fragment of the randomly sheared genomic DNA to produce an RNA/DNA duplex. The overhanging ends of the genomic fragment in the RNA/DNA duplex can then be enzymatically digested.

Claims

1. A method comprising:

(a) hybridizing randomly sheared genomic DNA to a halo probe to produce a first circular complex, wherein said halo probe comprises;

(i) a first oligonucleotide comprising flanking sequences that hybridize to different regions in a fragment of the randomly sheared genomic DNA and a central sequence; and

(ii) one or more second oligonucleotides that are complementary to the central sequence of the first oligonucleotide;

(b) enzymatically digesting the overhanging ends of the genomic fragment in the first circular complex to provide a second circular complex in which 5′ and

3′

ends of the one or more second oligonucleotide are ligatably adjacent to the 3′ and

5′

ends of the digested genomic fragment; and

(c) ligating the ends of the digested genomic fragment of (c) to the ends of the one or more second oligonucleotide to produce a circular DNA molecule.

2. The method of claim 1, wherein said method comprises:

(d) amplifying the digested genomic fragment from said circular DNA molecule using one or more primers that bind to sites that are provided by the one or more second oligonucleotides.

3. The method of claim 2, further comprising:

(e) sequencing the amplification product of (d) to provide the nucleotide sequence of at least part of the digested genomic fragment.

4. The method of any prior claim, wherein said first oligonucleotide comprises a capture moiety and wherein said method comprises, between steps (a) and (b), isolating said first circular complex using said capture moiety;

5. The method of claim 3, wherein said sequencing is done using primers that hybridize to sequencing primer sites in said one or more second oligonucleotides.

6. The method of any prior claim, wherein said enzymatically digesting comprises digesting said first circular complex using a single-strand specific bi-directional exonuclease, in the optional presence of a polymerase.

7. The method of any prior claim, wherein said single-strand specific bi-directional exonuclease is exonuclease VII.

8. The method of any prior claim, wherein said enzymatically digesting comprises treatment with a Pfu DNA polymerase/Taq DNA polymerase cocktail T4 DNA polymerase/exonuclease VII cocktail, treatment with a mung bean nuclease, treatment with a flap endonuclease in combination with another 3′

endonuclease.

9. The method of any prior claim, wherein the randomly sheared genomic DNA is produced from genomic DNA using chemical, physical or transposase-catalyzed fragmentation methods.

10. The method of claim 8, wherein said physical fragmentation methods comprise sonication, nebulization, or shearing.

11. The method of any prior claim, wherein said one or more second oligonucleotides is a single oligonucleotide that is complementary to the central sequence of the first oligonucleotide.

12. The method of any prior claim, wherein said one or more second oligonucleotides is two oligonucleotides, each comprises a first region that hybridizes to said first oligonucleotide, and a second region that provides binding sites for one or more amplification primers.

13. A method comprising:

(a) hybridizing randomly sheared genomic DNA to an RNA oligonucleotide comprising a region that hybridizes to a fragment of the randomly sheared genomic DNA to produce an RNA/DNA duplex;

(b) enzymatically digesting the overhanging ends of the genomic fragment in the RNA/DNA duplex to provide a duplex comprising a digested genomic fragment that has defined ends;

(c) digesting the RNA oligonucleotide of the duplex of (b) to release the digested genomic fragment;

(d) hybridizing the digested genomic fragment of (c) with a halo probe comprising;

(i) a first oligonucleotide comprising flanking sequences that hybridize to the ends of the digested genomic fragment and a central sequence; and

(ii) one or more second oligonucleotides that are complementary to the central sequence of the first oligonucleotide, to provide a second complex in which 5′ and

3′

ends of the second oligonucleotide are ligatably adjacent to the 3′ and

5′

ends of the digested genomic fragment;

(e) ligating the ends of the digested genomic fragment to the second oligonucleotide to produce a circular DNA molecule.

14. The method of claim 13, wherein said RNA oligonucleotide comprises a capture moiety and said method comprises isolating the enzymatically digested first complex of (b) using said capture moiety.

15. The method of claim 13 or 14, wherein said digesting step (c) is done using NaOH or RNAseH treatment.

16. The method of any of claims 13-15, wherein said method comprises:

(f) amplifying the digested genomic fragment from said circular DNA molecule using one or more primers that bind to sites that are in the central sequence of said first oligonucleotide.

17. The method of claim 16, further comprising:

(g) sequencing the amplification product of (f).

18. The method of claim 17, wherein said sequencing is done using primers that hybridize to sequencing primer sites that are provided by the one or more second oligonucleotides.

19. The method of any of claims 13-18, wherein said enzymatically digesting comprises digesting said first complex using exonuclease VII.

20. The method of any of claims 13-18, wherein said first probe comprises a capture moiety, and said method comprises isolating said second complex of (d) prior to said ligating step (e).