# US 2023/0054869 A1 · Methods and Compositions Employing Blocked Primers

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- Number: US 2023/0054869 A1
- Document ID: US20230054869A1
- Status: Published application
- Kind: A1
- Application number: 17/825,878
- Filed: 2022-05-26
- Published: 2023-02-23
- Earliest claimed: 2016-06-10
- Assignees: Takara Bio USA, Inc.
- Applicants: Takara Bio USA, Inc.
- Inventors: Thomas D. Schaal; Jude Menlo Dunne; Maithreyan Srinivasan; Alain Mir
- Web page: https://patentgrep.com/patent/US20230054869A1

## CPC

- C12Q 1/6853: using modified primers or templates
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6844: Nucleic acid amplification reactions
- 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
- 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

The present disclosure provides methods, compositions, and systems employing blocked primers. Aspects of the disclosure include providing a blocked primer reaction mixture that includes a blocked primer and a template nucleic acid component from a single cell; unblocking the blocked primer to produce an active primer reaction mixture and subjecting the activated primer reaction mixture to primer extension conditions, such as nucleic acid implication conditions.

## Claims

### Claim at position 1

1-15. (canceled)

### Claim 16

A method comprising:
(a) combining a blocked primer comprising a light labile blocking moiety with a template nucleic acid component to produce a blocked primer reaction mixture;
(b) unblocking the blocked primer of the blocked primer reaction mixture by exposing the blocked primer reaction mixture to light sufficient to remove the blocking moiety to produce an activated primer reaction mixture; and
(c) subjecting the activated primer reaction mixture to template dependent primer extension reaction conditions.

### Claim 17 (depends on 16)

The method according to claim 16, wherein the blocked primer is a member of a pair of blocked amplifications primers.

### Claim 18 (depends on 16)

The method according to claim 16, wherein the template dependent primer extension reaction conditions comprise nucleic acid amplification conditions.

### Claim 19 (depends on 16)

The method according to claim 16, wherein the template nucleic acid component comprises ribonucleic acids or deoxyribonucleic acids.

### Claim 20 (depends on 16)

The method according to claim 16, wherein the template nucleic acid component is produced by a polymerase mediated template dependent primer extension reaction.

### Claim 21 (depends on 20)

The method according to claim 20, wherein the polymerase mediated template dependent primer extension reaction comprises a reverse transcriptase mediated template dependent primer extension reaction.

### Claim 22 (depends on 16)

The method according to claim 16, wherein the template nucleic acid component is produced by obtaining a single cell and lysing the obtained single cell.

### Claim 23 (depends on 16)

The method according to claim 16, wherein the method comprises producing the blocked primer reaction mixture by introducing a single cell into a vessel comprising the blocked primer and lysing the introduced single cell.

### Claim 24 (depends on 16)

The method according to claim 16, wherein the method further comprises performing a second template mediated primer extension reaction.

### Claim 25 (depends on 24)

The method according to claim 24, wherein the second template mediated primer extension reaction is performed with an activated primer produced by unblocking a second blocked primer that is differentially blocked from the first blocked primer.

### Claim 26 (depends on 25)

The method according to claim 25, wherein the second blocked primer is a member of a second pair of blocked amplification primers and the method comprises unblocking the second pair of blocked amplification primers and then using the unblocked second pair of amplification primers in a second nucleic acid amplification.

### Claim 27 (depends on 16)

The method according to claim 16, wherein the light labile blocking moiety is attached to the 3′ end of the blocked primer.

### Claim 28 (depends on 16)

The method according to claim 16, wherein the blocked primer comprises non-template nucleotide sequence.

### Claim 29 (depends on 16)

The method according to claim 16, wherein the method is used in preparing a library selected from an immune cell receptor repertoire library, a T-cell receptor (TCR) repertoire library, a B-cell receptor (BCR) repertoire library, an expression library, a sequencing library, and a next-generation sequencing (NGS) library.

### Claim 30

A method comprising:
(a) combining a blocked primer comprising a thermally labile blocking moiety with a template nucleic acid component to produce a blocked primer reaction mixture;
(b) unblocking the blocked primer of the blocked primer reaction mixture by heating the blocked primer reaction mixture to a temperature sufficient to remove the thermally labile blocking moiety to produce an activated primer reaction mixture; and
(c) subjecting the activated primer reaction mixture to template dependent primer extension reaction conditions.

### Claim 31 (depends on 30)

The method according to claim 30, wherein the blocked primer is a member of a pair of blocked amplifications primers.

### Claim 32 (depends on 30)

The method according to claim 30, wherein the template nucleic acid component comprises ribonucleic acids or deoxyribonucleic acids.

### Claim 33 (depends on 30)

The method according to claim 30, wherein the template nucleic acid component is produced by a reverse transcriptase mediated template dependent primer extension reaction.

### Claim 34 (depends on 30)

The method according to claim 30, wherein the template nucleic acid component is produced by obtaining a single cell and lysing the obtained single cell.

### Claim 35 (depends on 30)

The method according to claim 30, wherein the blocked primer comprises non-template nucleotide sequence.

### Claim 36 (depends on 30)

The method according to claim 30, wherein the method is used in preparing a library selected from an immune cell receptor repertoire library, a T-cell receptor (TCR) repertoire library, a B-cell receptor (BCR) repertoire library, an expression library, a sequencing library, and a next-generation sequencing (NGS) library.

### Claim 37

A method comprising:
(a) combining a blocked primer comprising an enzymatically labile blocking moiety with a template nucleic acid component to produce a blocked primer reaction mixture;
(b) adding an enzyme to the blocked primer reaction mixture to enzymatically remove the enzymatically labile blocking moiety of the blocked primer, thereby unblocking the blocked primer of the blocked primer reaction mixture to produce an activated primer reaction mixture; and
(c) subjecting the activated primer reaction mixture to template dependent primer extension reaction conditions.

### Claim 38 (depends on 37)

The method according to claim 37, wherein the blocked primer is a member of a pair of blocked amplifications primers.

### Claim 39 (depends on 37)

The method according to claim 37, wherein the template nucleic acid component comprises ribonucleic acids or deoxyribonucleic acids.

### Claim 40 (depends on 37)

The method according to claim 37, wherein the template nucleic acid component is produced by a reverse transcriptase mediated template dependent primer extension reaction.

### Claim 41 (depends on 37)

The method according to claim 37, wherein the template nucleic acid component is produced by obtaining a single cell and lysing the obtained single cell.

### Claim 42 (depends on 37)

The method according to claim 37, wherein the blocked primer comprises non-template nucleotide sequence.

### Claim 43 (depends on 37)

The method according to claim 37, wherein the method is used in preparing a library selected from an immune cell receptor repertoire library, a T-cell receptor (TCR) repertoire library, a B-cell receptor (BCR) repertoire library, an expression library, a sequencing library, and a next-generation sequencing (NGS) library.

### Claim at position 30

44-47. (canceled)