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-12359254-B2
Application number	US-202017631418-A
Country	US
Kind code	B2
Filing date	Jul 31, 2020
Priority date	Jul 31, 2019
Publication date	Jul 15, 2025
Grant date	Jul 15, 2025

Abstract

where A 1 represents —S—, —S—S—, or —Se—, B represents a base, and R 1 represents a decomposable protecting 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; Klenow fragment for making the 3′ ends blunt; 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 A 1 represents —S—, —S—S—, or —Se—, B represents a base, a symbol * represents a bond to a sugar of an adjacent nucleotide, and R 1 is represented by:
a photodecomposable protecting group represented by the following formula (2A):

wherein A 2 represents an alkylene group having 1 to 3 carbon atoms, A 3 represents an alkylene group having 1 to 3 carbon atoms, and a symbol * represents a bond to A 1 ; or
a fluoride decomposable protecting group represented by the following formula (2B):

where A 4 represents an alkylene group having 1 to 3 carbon atoms, R 2 to R 4 each represent a straight or branched alkyl group having 1 to 4 carbon atoms, the R 2 to R 4 may be the same as or different from each other, and a symbol * represents a bond to A 1 .

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

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

4. A device for producing double-stranded DNA having sticky ends by using a primer for amplifying a nucleic acid, the device 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 the following formula (1):

where A 1 represents —S—, —S—S—, or —Se—, B represents a base, R 1 represents a decomposable protecting group, and a symbol * represents a bond to a sugar of an adjacent nucleotide;
a reverse primer being complementary to a part of a sequence of a sense strand of the template DNA and having the 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;
a blunting unit for making the 3′ ends of the double-stranded DNA blunt by Klenow fragment; and
a deprotection cleavage unit for deprotecting the R 1 , and cleaving DNA at the bond at the 3′-carbon of formula (1) to form a sticky end with a 3′-protruding end.

5. A method for producing double-stranded DNA having sticky ends by using a primer for amplifying a nucleic acid, the method 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 the following formula (1):

where A 1 represents —S—, —S—S—, or —Se—, B represents a base, R 1 represents a decomposable protecting group, and a symbol * represents a bond to a sugar of an adjacent nucleotide, and a reverse primer being complementary to a part of a sequence of a sense strand of the template DNA and having the 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;
blunting step of making the 3′ ends of the double-stranded DNA blunt by Klenow fragment; and
a deprotection cleavage step of deprotecting the R 1 , and cleaving DNA at the bond at the 3′-carbon of formula (1) to form a sticky end with a 3′-protruding end.

6. The method according to claim 5 , wherein
the R 1 is a photodecomposable protecting group represented by the following formula (2A):

where A 2 represents an alkylene group having 1 to 3 carbon atoms, A 3 represents an alkylene group having 1 to 3 carbon atoms, and a symbol * represents a bond to A 1 , and is deprotected by photoirradiation in the deprotection cleavage step.

7. The method according to claim 5 , wherein
the R 1 is a fluoride decomposable protecting group represented by the following formula (2B)

where A 4 represents an alkylene group having 1 to 3 carbon atoms, R 2 to R 4 each represent a straight or branched alkyl group having 1 to 4 carbon atoms, the R 2 to R 4 may be the same as or different from each other, and a symbol * represents a bond to A 1 , and is deprotected by a fluoride in the deprotection cleavage step.

8. The method according to claim 6 , wherein
the R 1 is a 2-nitrobenzyloxymethyl group represented by the following formula (3A):

9. The method according to claim 7 , wherein
the R 1 is a triisopropylsilyloxymethyl group represented by the following formula (3B):

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

where A 2 represents an alkylene group having 1 to 3 carbon atoms, A 3 represents an alkylene group having 1 to 3 carbon atoms, and a symbol * represents a bond to A 1 , and is deprotected by photoirradiation in the deprotection cleavage step.

11. The device according to claim 4 , wherein
the R 1 is a fluoride decomposable protecting group represented by the following formula (2B):

where A 4 represents an alkylene group having 1 to 3 carbon atoms, R 2 to R 4 each represent a straight or branched alkyl group having 1 to 4 carbon atoms, the R 2 to R 4 may be the same as or different from each other, and a symbol * represents a bond to A 1 , and is deprotected by a fluoride in the deprotection cleavage step.

12. The device according to claim 10 , wherein
the R 1 is a 2-nitrobenzyloxymethyl group represented by the following formula (3A):

13. The device according to claim 11 , wherein
the R 1 is a triisopropylsilyloxymethyl grou

Assignees

• Japan Science & Tech Agency

Inventors

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

Classifications

• Y02P20/55

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

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C07H1/02

  Phosphorylation · CPC title

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title

• C07H23/00

  Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12 (esters with inorganic acids C07H11/00) · CPC title