Compositions and methods for selection of nucleic acids

Patent metadata

Field	Value
Publication number	US-2020399690-A1
Application number	US-202016828741-A
Country	US
Kind code	A1
Filing date	Mar 24, 2020
Priority date	Nov 1, 2012
Publication date	Dec 24, 2020
Grant date	—

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.

First claim

1 .- 35 . (canceled)

36 . A method for enrichment of a target region in a nucleic acid sample comprising:
a. fragmenting the nucleic acid sample to generate a mixture of double-stranded fragments, where a minority of the double-stranded fragments in the mixture comprise the target region, and a majority of the double-stranded fragments in the mixture do not comprise the target region; and b. selectively degrading the majority of the double-stranded fragments in the mixture that do not comprise the target region in the presence of the minority of the double-stranded fragments that comprise the target region, wherein the minority of the double-stranded fragments that comprise the target region are protected from the degrading, thereby enriching the mixture for the double-stranded fragments that comprise the target region.

37 . The method of claim 36 , wherein type IIs restriction enzymes are used in the fragmenting to generate the double-stranded fragments, wherein the minority of the double-stranded fragments comprising the target region have known overhang sequences at both ends.

38 . The method of claim 360 , wherein ligation of two stem-loop adapters to the double-stranded fragments that comprise the target region protects them from being degraded.

39 . The method of claim 36 , further comprising exposing the double-stranded fragments that comprise the target region to a primer and polymerase enzyme to generate a polymerase complex, and exposing the polymerase complex to a capture-hook oligonucleotide attached to a magnetic bead to selectively capture the polymerase complex.

40 . The method of claim 39 , wherein the capture-hook oligonucleotide only captures an active polymerase complex by binding to a region of one of the stem-loop adapters that has been rendered single-stranded by the polymerase enzyme.

41 . The method of claim 38 , wherein a primer binding sequence is present within the stem-loop adapter.

42 . The method of claim 41 , 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.

43 . The method of claim 42 , further comprising exposing the polymerase complex to a capture-hook oligonucleotide attached to a magnetic bead to selectively capture the polymerase complex.

44 . The method of claim 43 , 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.

45 . The method of claim 36 , further comprising performing template-directed sequencing-by-synthesis on the enriched nucleic acid fragments of the enriched mixture.

46 . The method of claim 45 , wherein the template-directed sequencing-by-synthesis generates redundant sequence information from single molecules of the enriched nucleic acid fragments.

47 . The method of claim 38 , 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.

48 . The method of claim 36 , wherein a first endonuclease and a second endonuclease are used in the fragmenting to generate the double-stranded fragments.

49 . The method of claim 48 , wherein the first endonuclease and 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.

50 . The method of claim 49 , wherein at least one of the first endonuclease and second endonuclease are engineered endonucleases which are engineered zinc finger DNA-binding protein endonucleases.

51 . The method of claim 48 , wherein the first endonuclease, the second endonuclease, or both leave ends with single-stranded overhangs.

52 . The method of claim 48 , wherein the first endonuclease, the second endonuclease, or both leave blunt ends.

53 . The method of claim 48 , wherein the target region comprises repetitive sequences.

54 . The method of claim 53 , wherein the repetitive sequences are repeat sequences associated with a genetic disorder.

55 . The method of claim 36 , wherein multiple different target regions are enriched in the nucleic acid sample.

Assignees

• Pacific Biosciences California Inc

Inventors

• Pham Thang Tat
• Tsai Yu-Chih
• Korlach Jonas
• Clark Tyson A
• Turner Stephen

Classifications

• C12N15/1072 Primary

  Differential gene expression library synthesis, e.g. subtracted libraries, differential screening · CPC title

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title