# US 10,246,702 B1 · Compositions and methods for labeling target nucleic acid molecules

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- Number: US 10,246,702 B1
- Document ID: US10246702B1
- Status: Granted
- Kind: B1
- Application number: 15/051,064
- Filed: 2016-02-23
- Granted: 2019-04-02
- Earliest claimed: 2015-02-23
- Assignees: New England Biolabs, Inc.
- Applicants: Cynthia L. Richard; Brendan Galvin
- Inventors: Cynthia L. Richard; Brendan Galvin
- Web page: https://patentgrep.com/patent/US10246702B1

## CPC

- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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 herein are methods and compositions for labeling target nucleic acid molecules with molecular barcodes.

## Claims

### Claim 1

A method, comprising:
(a) hybridizing a primer to a library of polynucleotide molecules comprising, in 3′ to 5′ order, a first adapter, a sequence derived from genomic DNA, cDNA or RNA, and a second adapter, wherein the primer hybridizes to the first adaptor;
(b) extending the 3′ end of the hybridized primer to produce primer extension products;
(c) hybridizing the primer extension products with synthetic oligonucleotides, wherein:
(i) the synthetic oligonucleotides comprise, in 3′ to 5′ order, a non-extendable 3′ end, a hybridization sequence, and a variable barcode sequence; and
(ii) the hybridization sequence hybridizes to a 3′ terminal region of the sequence complementary to the second adapter; and
(d) incubating the hybridized primer extension products of step (b) with a polymerase such that the polymerase extends the 3′ end of the primer extension products to form a further extended primer extension product.

### Claim 2 (depends on 1)

The method according to claim 1, further comprising the step of denaturing the primer extension products from the polynucleotide molecules prior to step (c).

### Claim 3 (depends on 1)

The method according to claim 1, wherein the 5′ terminus of the primer is protected from exonuclease digestion and the method further comprises incubating the primer extension products and the polynucleotide molecules with a 5′ exonuclease to remove a 5′ terminal sequence from the polynucleotide molecules prior to step (c).

### Claim 4 (depends on 3)

The method according to claim 3, wherein the second adaptor contains modified nucleotides that allow only partial digestion by a 5′ exonuclease.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the modified nucleotides are internal phosphorothioate-containing nucleotides.

### Claim 6 (depends on 4)

The method according to claim 4, wherein the 5′ exonuclease removes 10-20 bases from an end of the second adaptor leaving a single strand overhang.

### Claim 7 (depends on 1)

The method according to claim 1, further comprising removing a 5′ terminal sequence from the polynucleotide molecules after step (b) and prior to step (c), leaving a 3′ overhang.

### Claim 8 (depends on 1)

The method according to claim 1, further comprising amplifying the further extended primer extension products produced in step (d).

### Claim 9 (depends on 1)

The method of claim 1, wherein extension of the primer extension products in step (d) adds the complement of the variable barcode sequence onto the primer extension products.