Compositions and methods for selection of nucleic acids

• US 10,640,818 B2
• Filed: 11/16/2016
• Issued: 05/05/2020
• Est. Priority Date: 01/31/2012
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Pham, Thang Tat, Tsai, Yu-Chih, Korlach, Jonas, Clark, Tyson A., Turner, Stephen
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US15/353,595
• Publication Number:
  US 20170145492A1
• Time in Patent Office: 1,266 Days
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6855 Ligating adaptors

Abstract

Methods are provided for reducing the complexity of a population of nucleic acids prior to performing an analysis of the nucleic acids, e.g., sequence analysis. The methods result in a subset of the initial population enriched for a target region, which is typically located within one or more target fragments. The methods are particularly useful for analyzing populations having a high degree of complexity, e.g., chromosomal-derived DNA, whole genomic DNA, or mRNA populations.

Claims

1. A method for enrichment of a target region in a nucleic acid sample comprising:

a) obtaining a nucleic acid sample comprising a mixture of double-stranded nucleic acid fragments, wherein a subset of fragments in the mixture comprises the target region;

b) digesting the nucleic acid sample with a first endonuclease and a second endonuclease to produce a population of double-stranded nucleic acid fragments, wherein the first endonuclease cleaves a particular nucleotide sequence that is upstream of the target region and the second endonuclease cleaves a particular nucleotide sequence that is downstream of the target region;

c) ligating stem-loop adapters to the population of double-stranded nucleic acid fragments to produce a ligated sample; and

d) treating the ligated sample with;

(i) one or more third endonucleases that do not cleave stem-loop ligated double-stranded nucleic acid fragments that include the target region, and (ii) one or more exonucleases that digest double-stranded nucleic acid fragments cleaved by the one or more third endonucleases, having one stem-loop adapter ligated thereto, or having no stem-loop adapter ligated thereto, thereby enriching for the target region in the nucleic acid sample.

2. The method of claim 1, wherein a primer binding sequence is present within the stem-loop adapter.

3. The method of claim 2, further comprising hybridizing a primer to the primer binding sequence in the enriched double-stranded nucleic acid fragments and exposing the primer-hybridized double-stranded nucleic acid fragments to a polymerase enzyme to generate a polymerase complex.

4. The method of claim 3, further comprising exposing the polymerase complex to a capture-hook oligonucleotide attached to a magnetic bead to selectively capture the polymerase complex.

5. The method of claim 4, wherein the capture-hook oligonucleotide only captures an active polymerase complex by binding to a region of the nucleic acid in the polymerase complex that has been rendered single-stranded by the polymerase enzyme.

6. The method of claim 1, further comprising performing template-directed sequencing-by-synthesis on the enriched nucleic acid fragments.

7. The method of claim 6, wherein the template-directed sequencing-by-synthesis generates redundant sequence information from single molecules of the enriched nucleic acid fragments.

8. The method of claim 1, further comprising isolating fragments having an approximate size of a double-stranded nucleic acid fragment comprising the target region generated by digestion with the first endonuclease and a second endonuclease prior to the ligating step.

9. The method of claim 1, wherein the first endonuclease and the second endonuclease are selected from the group consisting of:

a type II restriction endonuclease, a type IIs restriction endonuclease, an engineered endonuclease, and any combination thereof.

10. The method of claim 9, wherein the engineered endonuclease is an engineered zinc finger DNA-binding protein endonuclease.

11. The method of claim 9, wherein the first endonuclease and the second endonuclease are engineered endonucleases.

12. The method of claim 1, wherein the first endonuclease, the second endonuclease, or both leave ends with single-stranded overhangs.

13. The method of claim 12, wherein:

the first endonuclease leaves a first single-stranded overhang and the second endonuclease leaves a second single-stranded overhang, wherein the first single-stranded overhang and the second single-stranded overhang are different; and

the stem-loop adapters comprise first stem-loop adapters and second stem-loop adapters, wherein the first stem-loop adapters comprise a single-stranded overhang that is complementary to the first single-stranded overhang and the second stem-loop adapters comprise a single-stranded overhang that is complementary to the second single-stranded overhang.

14. The method of claim 1, wherein the first endonuclease, the second endonuclease, or both leave blunt ends.

15. The method of claim 1, wherein the target region comprises repetitive sequences.

16. The method of claim 15, wherein the repetitive sequences are repeat sequences associated with a genetic disorder.

17. The method of claim 16, wherein the genetic disorder is selected from the group consisting of:

fragile X syndrome, fragile X-associated tremor/ataxia syndrome (FXTAS), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson'"'"'s disease, autism, cancer, myotonic dystrophy, ataxia, epilepsy, and Huntington'"'"'s disease.

18. The method of claim 1, wherein multiple different target regions are enriched in the nucleic acid sample.