Nucleic acid amplification
Methods and compositions for the amplification of nucleic acids are disclosed. Amplification methods provided herein may be performed under isothermal conditions. Methods and compositions may include reagents such as restriction enzymes, polymerases, ligases, primers, and polynucleotide adaptors.
1. A method for amplifying a nucleic acid, comprising:
(A) reverse transcribing a single-stranded RNA to produce a linear double-stranded nucleic acid comprising two separate complementary strands;
(B) forming a circular strand comprising a first polynucleotide adaptor and a second polynucleotide adaptor and the two complementary strands, wherein each adaptor comprises a single nucleic acid strand which contains a nucleotide sequence of one strand of a restriction enzyme recognition sequence;
(C) annealing the circular strand of step (B) with a first polynucleotide primer and synthesizing an extension product of the first primer;
(D) annealing the extension product of the first primer of step (C) with a second polynucleotide primer and synthesizing an extension product of the second primer, wherein the extension product of the second primer is complementary to the extension product of the first primer;
(E) producing a new double-stranded nucleic acid comprising at least a portion of the extension product of the first primer and at least a portion of the extension product of the second primer and a full double-stranded restriction enzyme recognition sequence corresponding to the sequence of the one strand of the restriction enzyme recognition sequence of step (B);
(F) cleaving the new double-stranded nucleic acid from step (E) with a restriction enzyme; and
(G) forming two or more shorter double-stranded nucleic acids, at least two of which comprise at least a portion of a copy of the linear double-stranded nucleic acid of step (A), thereby amplifying the linear double-stranded nucleic acid;
wherein steps (A)-(G) are performed in a reaction mixture under isothermal conditions, and wherein the reaction mixture comprises:
(i) the single-stranded RNA;
(ii) a reverse transcriptase;
(iii) the first polynucleotide adaptor and the second polynucleotide adaptor;
(iv) the first polynucleotide primer and the second polynucleotide primer;
(v) a ligase;
(vi) a polymerase; and
(vii) the restriction enzyme.
2. The method of claim 1 , further comprising repeating steps (B)-(G) for one or more additional cycles, using a shorter double-stranded nucleic acid of step (G) of a first cycle as the linear double-stranded nucleic acid of step (A) of a second cycle.
3. The method of claim 1 , wherein at least two of the shorter double-stranded nucleic acids of step (G) contain a complete copy of the linear double-stranded nucleic acid of step (A).
4. The method of claim 1 , wherein the two polynucleotide adaptors contain the nucleotide sequence of one strand of a restriction enzyme recognition sequence corresponding to the same restriction enzyme.
5. The method of claim 1 , wherein at least one of the adaptors contains a nucleotide sequence comprising a 5′ region, a middle region, and a 3′ region, wherein the 5′ region and 3′ region of the sequence are complementary to each other and form a stem of a stem-loop structure, and wherein the stem contains a blunt end.
6. The method of claim 1 , wherein all steps of the method are performed at a temperature of no greater than 70° C.
7. The method of claim 1 , wherein the linear double-stranded nucleic acid template is amplified at least 10-fold within 60 minutes of initiation of the method.