Nucleic acid amplification method
First Claim
1. A method for amplifying a nucleic acid, comprising the steps of:
- (a) ligating a double-stranded adapter that contains adapter DNA strands capable of forming a folded structure to a double-stranded DNA that contains a target DNA sequence to prepare a cyclic DNA template composed of nick-containing double-stranded DNA; and
(b) performing a 3′
end elongation reaction from the nick as an origin using a strand-displacement DNA polymerase, thereby producing a concatemer in which a plurality of the target DNA sequences and the adapter DNA strands capable of forming the folded structure are linked in series as a single-stranded DNA, wherein no primers other than said adapter are used in the method,wherein the concatemer has a folded shape due to the folded structure, andwherein the double-stranded adapter has an adapter DNA strand that has a first DNA sequence, a second DNA sequence, and a third DNA sequence, wherein the first and the third DNA sequences are capable of forming a folded structure, and the adapter DNA strand and an adapter DNA strand complementary thereto are bound to form a double-strand.
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Abstract
A nucleic acid amplification method includes ligating a double-stranded adapter (20) containing adapter DNA strands capable of forming a folded structure to a double-stranded DNA (1, 2) containing a target DNA sequence (1) to prepare a cyclic DNA template composed of double-stranded DNA containing a nick (5). A 3′-end elongation reaction is performed using a strand-displacement DNA polymerase from the nick (5) as an origin, thereby producing a concatemer (29) in which a plurality of the target DNA sequences (1) and the adapter DNA strands capable of forming the folded structure are linked in series as a single-stranded DNA. The concatemer (29) contains a plurality of the target DNA sequences (1) suitable for nucleotide sequence analysis and has a folded shape such that it takes the form of a ball due to its folded structure.
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Citations
5 Claims
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1. A method for amplifying a nucleic acid, comprising the steps of:
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(a) ligating a double-stranded adapter that contains adapter DNA strands capable of forming a folded structure to a double-stranded DNA that contains a target DNA sequence to prepare a cyclic DNA template composed of nick-containing double-stranded DNA; and (b) performing a 3′
end elongation reaction from the nick as an origin using a strand-displacement DNA polymerase, thereby producing a concatemer in which a plurality of the target DNA sequences and the adapter DNA strands capable of forming the folded structure are linked in series as a single-stranded DNA, wherein no primers other than said adapter are used in the method,wherein the concatemer has a folded shape due to the folded structure, and wherein the double-stranded adapter has an adapter DNA strand that has a first DNA sequence, a second DNA sequence, and a third DNA sequence, wherein the first and the third DNA sequences are capable of forming a folded structure, and the adapter DNA strand and an adapter DNA strand complementary thereto are bound to form a double-strand. - View Dependent Claims (2, 3, 4, 5)
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Specification