# US 2019/0169679 A1 · One-step reverse transcription template-switching pcr

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- Number: US 2019/0169679 A1
- Document ID: US20190169679A1
- Status: Published application
- Kind: A1
- Application number: 16/309,884
- Filed: 2017-06-23
- Published: 2019-06-06
- Earliest claimed: 2016-06-23
- Assignees: Riken
- Applicants: Riken
- Inventors: Katsuyuki Shiroguchi
- Web page: https://patentgrep.com/patent/US20190169679A1

## CPC

- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12N 15/09: Recombinant DNA-technology
- C12Q 1/68: involving nucleic acids
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6853: using modified primers or templates
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 2525/173: incorporating a polynucleotide run, e.g. polyAs, polyTs
- C12Q 2525/186: incorporating a non-extendable or blocking moiety
- 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

The present invention provides technology for carrying out one-step reverse transcription template-switching PCR more quickly, more easily, and with high specificity. The present invention provides a nucleic acid amplification method for amplifying at least a partial region of RNA using a modified oligonucleotide primer, said nucleic acid amplification method being characterized by the following: a nucleic acid amplification reaction comprises a reverse transcription step a) in which RNA is used as a template, a template switching step b) in which a template-switching oligonucleotide is added to cDNA synthesized in step a), and a DNA amplification step c) in which DNA amplification is carried out by PCR in which the template-switch cDNA synthesized in step b) is used as a template; the steps a) to c) are performed in a single stage in the same reaction system; as a result of being modified, some or all of the primer function of the modified oligonucleotide primer is blocked in the reverse transcription step a); and blocking of the primer function is cancelled in the DNA amplification step c).

## Claims

### Claim 1

A method of amplifying at least a part of a region of a target RNA, the method comprising the steps of:
a) mixing the target RNA, a reagent required for reverse transcription, a reagent required for template switching, and a reagent required for a polymerase chain reaction and subjecting the mixture to a condition under which reverse transcription occurs to provide a cDNA comprising a nucleic acid sequence corresponding to the target RNA and a template switching oligonucleotide; and
b) subjecting the cDNA obtained in step a) to a condition under which a polymerase chain reaction occurs to amplify at least a part of a region of the cDNA;
wherein the reagent required for a polymerase chain reaction comprises a modified oligonucleotide primer designed to have a primer function that is partially or completely blocked in step a) and designed to have blocking of the primer function cleared in step b).

### Claim 2

A method of producing a nucleic acid sample that is amplified based on at least a part of a region of a target RNA, the method comprising the steps of:
a) mixing the target RNA, a reagent required for reverse transcription, a reagent required for template switching, and a reagent required for a polymerase chain reaction and subjecting the mixture to a condition under which reverse transcription occurs to provide a cDNA comprising a nucleic acid sequence corresponding to the target RNA and a template switching oligonucleotide; and
b) subjecting the cDNA obtained in step a) to a condition under which a polymerase chain reaction occurs;
wherein the reagent required for a polymerase chain reaction comprises a modified oligonucleotide primer designed to have a primer function that is partially or completely blocked in step a) and designed to have blocking of the primer function cleared in step b).

### Claim 3 (depends on 1)

The method of claim 1, wherein the reagent required for a polymerase chain reaction optionally comprises a 5′ anchor oligonucleotide primer comprising at least a part of an anchor sequence comprised in the template switching oligonucleotide, optionally wherein the reagent required for a polymerase chain reaction does not comprise the 5′ anchor oligonucleotide primer.

### Claim 4

(canceled)

### Claim 5 (depends on 1)

The method of claim 1, wherein the reagent required for reverse transcription comprises an oligonucleotide primer that initiates reverse transcription, and the oligonucleotide primer that initiates reverse transcription is comprised in the mixture at a final concentration of about 40 nM or less, or at a mole ratio of about 1:10 or less relative to the modified oligonucleotide primer.

### Claim 6 (depends on 1)

The method of claim 1, wherein the modified oligonucleotide primer has one or more complementary regions on a sequence of the same modified oligonucleotide primer, and has a turn structure by the complementary regions or comprises a thermolabile modifying group before initial thermal denaturation of PCR.

### Claim 7 (depends on 1)

The method of claim 1, wherein the modified oligonucleotide primer comprises a nucleotide sequence that is complementary to a partial sequence of a template RNA, optionally wherein a part of the modified oligonucleotide primer whose primer function has not been blocked functions as an oligonucleotide primer that initiates reverse transcription by hybridizing to the template RNA.

### Claim 8

(canceled)

### Claim 9

A kit for amplifying at least a part of a region of a target RNA, the kit comprising:
i) a reagent required for reverse transcription;
ii) a reagent required for template switching;
iii) a reagent required for a polymerase chain reaction using a modified oligonucleotide primer; and
iv) optionally a user manual;
characterized in that the reagents of i) to iii) and the modified oligonucleotide primer are all mixed in a reaction system as of the initiation of a reaction, wherein the modified oligonucleotide primer is designed to have a primer function that is partially or completely blocked under a condition where reverse transcription occurs and designed to have blocking of the primer function cleared under a condition where a polymerase chain reaction occurs.

### Claim 10 (depends on 9)

The kit of claim 9, wherein the reagent required for template switching comprises a template switching oligonucleotide, and the reagent required for a polymerase chain reaction optionally comprises a 5′ anchor oligonucleotide primer comprising at least a part of an anchor sequence comprised in the template switching oligonucleotide, optionally wherein the reagent required for a polymerase chain reaction does not comprise the 5′ anchor oligonucleotide primer.

### Claim 11

(canceled)

### Claim 12 (depends on 9)

The kit of claim 9, characterized in that the reagent required for reverse transcription comprises an oligonucleotide primer that initiates reverse transcription, and the oligonucleotide primer that initiates reverse transcription is used at a final concentration of about 40 nM or less, or at a mole ratio of about 1:10 or less relative to the modified oligonucleotide primer.

### Claim 13 (depends on 9)

The kit of claim 9, wherein the modified oligonucleotide primer has one or more complementary regions on a sequence of the same modified oligonucleotide primer, and has a turn structure by the complementary regions or comprises a thermolabile modifying group before initial thermal denaturation of PCR.

### Claim 14 (depends on 9)

The kit of claim 9, wherein the modified oligonucleotide primer comprises a nucleotide sequence that is complementary to a partial sequence of a template RNA, optionally wherein a part of the modified oligonucleotide whose primer function has not been blocked functions as an oligonucleotide primer that initiates reverse transcription by hybridizing to the template RNA.

### Claim 15

(canceled)

### Claim 16

A composition for amplifying at least a part of a region of a target RNA, comprising a modified oligonucleotide primer, wherein the modified oligonucleotide primer is designed to have a primer function that is partially blocked under a condition where reverse transcription occurs and designed to have the blocking of the primer function cleared under a condition where a polymerase chain reaction occurs, wherein a part of the modified oligonucleotide primer whose primer function has not been blocked functions as an oligonucleotide primer that initiates reverse transcription by hybridizing to a template RNA.

### Claim 17 (depends on 16)

The composition of claim 16, wherein the modified oligonucleotide primer has one or more complementary regions on a sequence of the same modified oligonucleotide primer, and has a turn structure by the complementary regions or comprises a thermolabile modifying group before initial thermal denaturation of PCR.

### Claim 18 (depends on 16)

The composition of claim 16, wherein the composition is used in one-step reverse transcription template switching PCR.

### Claim 19 (depends on 2)

The method of claim 2, wherein the reagent required for a polymerase chain reaction optionally comprises a 5′ anchor oligonucleotide primer comprising at least a part of an anchor sequence comprised in the template switching oligonucleotide, optionally wherein the reagent required for a polymerase chain reaction does not comprise the 5′ anchor oligonucleotide primer.

### Claim 20 (depends on 2)

The method of claim 2, wherein the reagent required for reverse transcription comprises an oligonucleotide primer that initiates reverse transcription, and the oligonucleotide primer that initiates reverse transcription is comprised in the mixture at a final concentration of about 40 nM or less, or at a mole ratio of about 1:10 or less relative to the modified oligonucleotide primer.

### Claim 21 (depends on 2)

The method of claim 2, wherein the modified oligonucleotide primer has one or more complementary regions on a sequence of the same modified oligonucleotide primer, and has a turn structure by the complementary regions or comprises a thermolabile modifying group before initial thermal denaturation of PCR.

### Claim 22 (depends on 2)

The method of claim 2, wherein the modified oligonucleotide primer comprises a nucleotide sequence that is complementary to a partial sequence of a template RNA, optionally wherein a part of the modified oligonucleotide primer whose primer function has not been blocked functions as an oligonucleotide primer that initiates reverse transcription by hybridizing to the template RNA.