# US 2020/0399690 A1 · Compositions and methods for selection of nucleic acids

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- Number: US 2020/0399690 A1
- Document ID: US20200399690A1
- Status: Published application
- Kind: A1
- Application number: 16/828,741
- Filed: 2020-03-24
- Published: 2020-12-24
- Earliest claimed: 2012-11-01
- Assignees: Pacific Biosciences of California, Inc.
- Applicants: Pacific Biosciences of California, Inc.
- Inventors: Thang Tat Pham; Yu-Chih Tsai; Jonas Korlach; Tyson A. Clark; Stephen Turner
- Web page: https://patentgrep.com/patent/US20200399690A1

## CPC

- C12Q 1/6855: Ligating adaptors
- C12N 15/1072: {Differential gene expression library synthesis, e.g. subtracted libraries, differential screening}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## 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

### Claim at position 1

-35. (canceled)

### Claim 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.

### Claim 37 (depends on 36)

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.

### Claim 38 (depends on 360)

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.

### Claim 39 (depends on 36)

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.

### Claim 40 (depends on 39)

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.

### Claim 41 (depends on 38)

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

### Claim 42 (depends on 41)

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.

### Claim 43 (depends on 42)

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.

### Claim 44 (depends on 43)

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.

### Claim 45 (depends on 36)

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

### Claim 46 (depends on 45)

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.

### Claim 47 (depends on 38)

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.

### Claim 48 (depends on 36)

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

### Claim 49 (depends on 48)

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.

### Claim 50 (depends on 49)

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.

### Claim 51 (depends on 48)

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

### Claim 52 (depends on 48)

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

### Claim 53 (depends on 48)

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

### Claim 54 (depends on 53)

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

### Claim 55 (depends on 36)

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