| CPC C12P 19/34 (2013.01) [C12N 15/115 (2013.01); C12Q 1/6844 (2013.01); C12N 2310/16 (2013.01)] | 19 Claims |
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1. A method for producing a target single-stranded DNA, comprising:
(1) obtaining a template double-stranded DNA molecule consisting of a first strand and a reverse complementary second strand, wherein the sequence structure of the first strand is as shown in Formula (I):
5′ left aptamer sequence-target single-stranded DNA sequence-right aptamer sequence 3′ (I)
wherein:
the left aptamer sequence has a sequence structure of Formula: XnTXqXA; and the right aptamer sequence has a sequence structure of Formula: XBXq′Xn-m,
in which Xn is a nucleotide sequence consisting of n nucleotides, where the 5′-terminal nucleotide is A and n is any integer of at least 4;
Xq is the sequence of a type II restriction endonuclease recognition site, and Xq is not present in the target single-stranded DNA sequence; Xq′ is a reverse complementary sequence of Xq; XA and XB are 0 to several nucleotides, so that XqXA and XBXq′ constitute the cleavage site of the type II restriction endonuclease respectively, to allow the type II restriction endonuclease to cleave at the 3′ terminus of the XqXA sequence and the 5′ terminus of the XB Xq′ sequence;
Xn-m is a sequence of n-m nucleotides from the 5′ terminus of Xn, and m is an integer of 1-3; and
A represents the adenine nucleotide; and T represents the thymine nucleotide;
(2) with the template double-stranded DNA molecule as a template, performing PCR amplification using a forward primer and a reverse primer to obtain a product double-stranded DNA molecule comprising a first strand containing the target single-stranded DNA sequence and a reverse complementary second strand thereof;
wherein the forward primer comprises a sequence of XnUXqXA, and the reverse primer comprises a sequence of Xn-m′XqXB′, in which X1, Xq, XA, and XB are the same as in (1); Xn-m′ is a reverse complementary sequence of Xn-m, with the T at the 3′ terminus being replaced by U; XB′ is a reverse complementary sequence of XB; and U is the uracil nucleotide;
(3) cleaving the product double-stranded DNA molecule at U contained in the two strands with a uracil-specific excision reagent to produce cohesive terminuses at the two ends of the product double-stranded DNA molecule;
(4) ligating the two cohesive terminuses of the product double-stranded DNA molecule in the presence of a ligase, to form a circular double-stranded DNA having a gap in the first strand;
(5) subjecting the gapped circular double-stranded DNA obtained in Step (4) to rolling circle replication, where the replication starts with the gap in the sense first strand of the product double-stranded DNA molecule and uses the second strand as the template, to obtain a replicon comprising multiple sequence structures as shown in the following formula (II) in tandem:
5′-XnTXqXA-target single-stranded DNA sequence-XBXq′-3′ (II)
(6) annealing the replicon, so that a hairpin structure is formed between two adjacent target single-stranded DNA sequences in the replicon, wherein the hairpin structure consists of a sequence of Xq′ XnTXq;
(7) treating the replicon with the type II restriction endonuclease, and cleaving at the 5′ terminus and 3 terminus of the XBXq′ XnTXqXA sequence between adjacent target single-stranded DNA sequences to release multiple target single-stranded DNA sequences.
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