Primer, device for producing double-stranded DNA using primer, and method for producing double-stranded DNA using primer

Patent metadata

Field	Value
Publication number	US-12448637-B2
Application number	US-202017631407-A
Country	US
Kind code	B2
Filing date	Jul 31, 2020
Priority date	Jul 31, 2019
Publication date	Oct 21, 2025
Grant date	Oct 21, 2025

Abstract

A primer for amplifying a nucleic acid having a structure represented by the formula (1):

where B represents a base, R 1 represents a decomposable protecting group, and R 2 represents hydrogen or a hydroxyl group, and the symbol * represents a bond to a sugar of an adjacent nucleotide. A device for producing double-stranded DNA, includes: a forward primer and a reverse primer, having a structure represented by formula (1); a PCR device for forming double-stranded DNA with 3′-recessed ends by performing multiple cycles of PCR by using a template DNA as a template; and a photoirradiation unit for deprotecting R 1 and forming a sticky end with a 3′-protruding end.

First claim

The invention claimed is:

1. A primer for amplifying a nucleic acid, having a structure represented by the following formula (1):

where B represents a base, R 1 represents a decomposable protecting group, R 2 represents hydrogen or a hydroxyl group, and a symbol * represents a bond to a sugar of an adjacent nucleotide,
wherein the R 1 is a photodecomposable protecting group represented by the following formula (2A) or a reducing agent-decomposable protecting group represented by the following formula (2B):

where A 1 represents an alkylene group having 1 to 3 carbon atoms and may have a branched chain having 1 to 20 carbon atoms, and a symbol * of formula (2A) represents a bond to oxygen (O) of phosphoric acid,
where A 2 represents an alkylene group having 1 to 3 carbon atoms and may have a branched chain having 1 to 20 carbon atoms, and a symbol * of formula (2B) represents a bond to oxygen (O) of phosphoric acid.

2. The primer according to claim 1 , wherein the R 1 is a photodecomposable protecting group represented by the following formula (4A):

where R 3 represents an alkyl group having 1 to 20 carbon atoms.

3. The primer according to claim 2 , wherein the R 3 is a tert-butyl group or an adamantyl group.

4. The primer according to claim 1 , wherein the R 1 is a 2-nitrobenzyl group represented by the following formula (3A):

5. The primer according to claim 1 , wherein the R 1 is a 4-nitrobenzyl group represented by the following formula (3B):

6. The primer according to claim 1 , wherein two or more structures each represented by the formula (1) are continuous in a sequence.

7. A device for producing double-stranded DNA having sticky ends by using the primer according to claim 1 , comprising:
a forward primer being complementary to a part of a sequence of an antisense strand of a template DNA to be used as a template and having a structure represented by formula (1);
a reverse primer being complementary to a part of a sequence of a sense strand of the template DNA and having a structure represented by formula (1);
an amplification unit for performing a plurality of cycles of polymerase chain reaction (PCR) by using the template DNA as a template to form a forward-side extended chain being the forward primer extended and a reverse-side extended chain being the reverse primer extended, and for annealing the forward-side extended chain and the reverse-side extended chain to form double-stranded DNA with 3′-recessed ends; and
a deprotection unit for deprotecting the R 1 .

8. A method for producing double-stranded DNA having sticky ends by using the primer according to claim 1 , comprising:
a preparation step of preparing a forward primer being complementary to a part of a sequence of an antisense strand of a template DNA to be used as a template and having a structure represented by formula (1), and a reverse primer being complementary to a part of a sequence of a sense strand of the template DNA and having a structure represented by formula (1);
an amplification step of performing a plurality of cycles of polymerase chain reaction (PCR) by using the template DNA as a template to form a forward-side extended chain being the forward primer extended and a reverse-side extended chain being the reverse primer extended, and of annealing the forward-side extended chain and the reverse-side extended chain to form double-stranded DNA with 3′-recessed ends; and
a deprotection step of deprotecting the R 1 .

9. The method according to claim 8 , wherein the primer has a sequence in which two or more structures each represented by the formula (1) are continuous.

Assignees

• Japan Science & Tech Agency

Inventors

• Abe Hiroshi
• Abe Naoko
• Nakamoto Kosuke
• Murase Hiroki
• Kimura Yasuaki

Classifications

• C07H19/20

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• Y02P20/55

  Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title