# US 10,767,220 B2 · Methods of amplifying nucleic acids and compositions for practicing the same

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- Number: US 10,767,220 B2
- Document ID: US10767220B2
- Status: Granted
- Kind: B2
- Application number: 15/563,939
- Filed: 2016-05-17
- Published: 2018-05-10
- Granted: 2020-09-08
- Earliest claimed: 2015-05-21
- Assignees: Becton, Dickinson and Company
- Applicants: Becton, Dickinson and Company
- Inventors: Kristen Mary Borchert; Frances Poyen Tong; Charlotte Ann Brown; Richard Lee Kelley; Chang Chen; Jeffrey Propse Baker; Liwen Stacy Xu
- Web page: https://patentgrep.com/patent/US10767220B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2535/122: Massive parallel sequencing
- C12Q 2537/143: Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
- 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

## Abstract

Provided are methods of amplifying nucleic acids. The methods include combining a nucleic acid sample and one or more amplification primers adapted to amplify a region of one or more copy number stable genes in a reaction mixture under conditions sufficient to amplify the one or more copy number stable genes. Aspects of the present disclosure further include compositions and kits that find use in practicing embodiments of the methods.

## Claims

### Claim 1

A method of amplifying nucleic acids, comprising:
combining:
a nucleic acid sample; and
amplification primers adapted to amplify a region of six or more copy number stable genes selected from a group consisting of: HNRNPR, TCEB3, IL22RA1, RCAN3, GJB5, SLC25A44, MT3, MT1X, NUP93, RABL2B,
in a reaction mixture under conditions sufficient to amplify the six or more copy number stable genes.

### Claim 2 (depends on 1)

The method according to claim 1, further comprising amplifying one or more nucleic acids of interest present in the nucleic acid sample.

### Claim 3 (depends on 2)

The method according to claim 2, further comprising sequencing the amplified one or more copy number stable genes and the amplified one or more nucleic acids of interest.

### Claim 4 (depends on 3)

The method according to claim 3, further comprising determining the copy number of the one or more nucleic acids of interest present in the nucleic acid sample based on:
the number of sequencing reads corresponding to the one or more nucleic acids of interest present in the nucleic acid sample; and
the number of sequencing reads corresponding to the one or more copy number stable genes.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the amplification primers are adapted to amplify a region of 8 or more copy number stable genes selected from the group consisting of: HNRNPR, TCEB3, IL22RA1, RCAN3, GJB5, SLC25A44, MT3, MT1X, NUP93, and RABL2.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the amplification primers are adapted to amplify a region of each of the copy number stable genes selected from the group consisting of: HNRNPR, TCEB3, IL22RA1, RCAN3, GJB5, SLC25A44, MT3, MT1X, NUP93, and RABL2B.

### Claim 7

A method of amplifying nucleic acids, comprising:
combining:
a nucleic acid sample; and
primers adapted to amplify a region of each of the copy number stable genes selected from the group consisting of: SLC25A44, MT3, MT1X, NUP93, and RABL2B.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the combining comprises combining a known amount of nucleic acids corresponding to six or more copy number stable genes with the nucleic acid sample and the amplification primers adapted to amplify a region of six or more copy number stable genes.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the nucleic acid sample comprises nucleic acids isolated from one or more cells of a cellular sample of interest.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the cellular sample of interest is a single cell.

### Claim 11 (depends on 9)

The method according to claim 9, wherein the cellular sample of interest is a tumor sample.

### Claim 12 (depends on 4)

The method according to claim 4, wherein determining the copy number of the one or more nucleic acids of interest present in the nucleic acid sample is based on a ratio of the number of sequencing reads corresponding to the one or more nucleic acids of interest present in the nucleic acid sample to the number of sequencing reads corresponding to the one or more copy number stable genes.

### Claim 13 (depends on 7)

The method according to claim 7, further comprising amplifying one or more nucleic acids of interest present in the nucleic acid sample.

### Claim 14 (depends on 13)

The method according to claim 13, further comprising sequencing the amplified one or more copy number stable genes and the amplified one or more nucleic acids of interest.

### Claim 15 (depends on 14)

The method according to claim 14, further comprising determining the copy number of the one or more nucleic acids of interest present in the nucleic acid sample based on:
the number of sequencing reads corresponding to the one or more nucleic acids of interest present in the nucleic acid sample; and
the number of sequencing reads corresponding to the one or more copy number stable genes.

### Claim 16 (depends on 7)

The method according to claim 7, wherein the combining comprises combining a known amount of nucleic acids corresponding to the copy number stable genes with the nucleic acid sample and the amplification primers adapted to amplify a region of the copy number stable genes.

### Claim 17 (depends on 7)

The method according to claim 7, wherein the nucleic acid sample comprises nucleic acids isolated from one or more cells of a cellular sample of interest.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the cellular sample of interest is a single cell.

### Claim 19 (depends on 17)

The method according to claim 17, wherein the cellular sample of interest is a tumor sample.

### Claim 20 (depends on 15)

The method according to claim 15, wherein determining the copy number of the one or more nucleic acids of interest present in the nucleic acid sample is based on a ratio of the number of sequencing reads corresponding to the one or more nucleic acids of interest present in the nucleic acid sample to the number of sequencing reads corresponding to the copy number stable genes.