Primer, device for producing double-stranded DNA using primer, and method for producing double-stranded DNA using primer
Inventors
Abe, Hiroshi • Abe, Naoko • Nakamoto, Kosuke • Murase, Hiroki • Kimura, Yasuaki
Assignees
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Publication Number
US-12359254-B2
Publication Date
2025-07-15
Expiration Date
Abstract
where A1 represents —S—, —S—S—, or —Se—, B represents a base, and R1 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 R1 and forming a sticky end with a 3′-protruding end.
Core Innovation
The invention relates to a primer for amplifying a nucleic acid, where the primer has a structure represented by formula (1) and includes a decomposable protecting group R1. In formula (1), A1 represents —S—, —S—S—S—, or —Se—, B represents a base, and * represents a bond to a sugar of an adjacent nucleotide. The decomposable protecting group R1 is represented by either a photodecomposable protecting group formula (2A) or a fluoride decomposable protecting group formula (2B).
A device and a method are provided for producing double-stranded DNA having sticky ends by using the primer for amplifying a nucleic acid. A template DNA is amplified by performing cycles of polymerase chain reaction (PCR) using forward and reverse primers having formula (1), to form forward-side extended and reverse-side extended chains that are annealed to form double-stranded DNA with 3′-recessed ends. The 3′ ends are blunted by Klenow fragment, and then R1 is deprotected and DNA is cleaved at a bond at the 3′-carbon of formula (1) to form a sticky end with a 3′-protruding end.
The photodecomposable protecting group (formula (2A)) is deprotected by photoirradiation and the fluoride decomposable protecting group (formula (2B)) is decomposed by fluoride treatment. Sticky ends can be designed for cloning and library preparation without restriction enzymes, and the produced DNA is usable for genome build-up reactions including in vitro genome build-up and in-cell genome build-up.
Claims Coverage
The independent claims are clm-00001, clm-00004, and clm-00005. Across these claims, the coverage centers on a formula (1) primer with a decomposable protecting group R1 and on a PCR-plus-processing device/method that yields double-stranded DNA with 3′-protruding sticky ends via blunting and R1 deprotection/cleavage at the 3′-carbon of formula (1).
Primer for amplifying a nucleic acid with formula (1) and decomposable protecting group R1
A primer for amplifying a nucleic acid having a structure represented by formula (1), where A1 represents —S—, —S—S—S—, or —Se—, B represents a base, * represents a bond to a sugar of an adjacent nucleotide, and R1 is represented by either a photodecomposable protecting group represented by formula (2A) or a fluoride decomposable protecting group represented by formula (2B).
Device for producing double-stranded DNA with sticky ends using formula (1) primers and R1 cleavage
A device for producing double-stranded DNA having sticky ends by using a primer for amplifying a nucleic acid, including a forward primer and a reverse primer each having a structure represented by formula (1) with R1 as a decomposable protecting group; an amplification unit performing a plurality of cycles of PCR to form double-stranded DNA with 3′-recessed ends by annealing forward-side and reverse-side extended chains; a blunting unit for making the 3′ ends blunt by Klenow fragment; and a deprotection cleavage unit for deprotecting R1 and cleaving DNA at the bond at the 3′-carbon of formula (1) to form a sticky end with a 3′-protruding end.
Method for producing double-stranded DNA with sticky ends using formula (1) primers and R1 cleavage
A method for producing double-stranded DNA having sticky ends by using a primer for amplifying a nucleic acid, comprising a preparation step preparing a forward primer and a reverse primer each having a structure represented by formula (1) with R1 as a decomposable protecting group; an amplification step performing a plurality of cycles of PCR to form double-stranded DNA with 3′-recessed ends by annealing forward-side and reverse-side extended chains; a blunting step making the 3′ ends blunt by Klenow fragment; and a deprotection cleavage step deprotecting R1 and cleaving DNA at the bond at the 3′-carbon of formula (1) to form a sticky end with a 3′-protruding end.
The claims cover a formula (1) primer whose R1 is either photodecomposable (formula (2A)) or fluoride-decomposable (formula (2B)), and a device and method that amplify to obtain 3′-recessed ends, blunt with Klenow fragment, and then deprotect/cleave at the 3′-carbon of formula (1) to generate 3′-protruding sticky ends.
Stated Advantages
Site specificity.
Low cost.
Sticky ends can be designed without restriction enzymes for cloning, library preparation, and genome build-up reactions.
Documented Applications
Producing double-stranded DNA having sticky ends by using a primer for amplifying a nucleic acid.
Cloning using restriction enzyme-free sticky ends generated by the disclosed primer/device/method.
Library preparation using restriction enzyme-free sticky ends generated by the disclosed primer/device/method.
Genome build-up reactions, including in vitro genome build-up.
Genome build-up reactions, including in-cell genome build-up.
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