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 linear double-stranded nucleic acid template comprising two separate complementary strands, comprising:
(A) ligating an adaptor comprising a single nucleic acid strand to each end of a linear double-stranded nucleic acid template, to yield a circular strand containing the general formula in the 5′ to 3′ direction: -A1-S1-A2-S2-, wherein
each adaptor contains at least four nucleotide bases and a single strand component of a restriction enzyme recognition sequence,
A1 and A2 denote separate adaptors,
S1 denotes a first complementary strand of the linear double-stranded nucleic acid template,
S2 denotes a second complementary strand of the linear double-stranded nucleic acid template,
the 3′ terminus of A1 is linked to the 5′ terminus of S1,
the 3′ terminus of S1 is linked to the 5′ terminus of A2,
the 3′ terminus of A2 is linked to the 5′ terminus of S2, and
the 3′ terminus of S2 is linked to the 5′ terminus of A1;
(B) annealing a first oligonucleotide primer to the circular strand;
(C) extending the first oligonucleotide primer along the circular strand by using a polymerase, to form an extension product of the first primer;
(D) annealing a second oligonucleotide primer to the extension product of the first primer;
(E) extending the second oligonucleotide primer along at least a portion of the extension product of the first primer by using a polymerase, to produce 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;
(F) cleaving the new double-stranded nucleic acid of step with a restriction enzyme that recognizes a full double-stranded restriction enzyme recognition sequence corresponding to the single strand component of a restriction enzyme sequence of at least one of the adaptors, to form 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 template of step (A), thereby amplifying the linear double-stranded nucleic acid template; and
(G) repeating steps (A)-(F) for one or more additional cycles, using the double stranded nucleic acid of step (F) of a first cycle as the linear double-stranded nucleic acid template of step (A) of a second cycle.
2. The method of claim 1 , wherein at least two of the shorter double-stranded nucleic acids contain a complete copy of the linear double-stranded nucleic acid template of step (A).
3. The method of claim 1 , further comprising wherein steps (B) and (C) of the second cycle comprises treating at least two different circular strands with the first oligonucleotide primer and polymerase, wherein the at least two different circular strands comprise: i) a circular strand formed in the first cycle and ii) a circular strand formed in the second cycle.
4. The method of claim 1 , wherein the first oligonucleotide primer is complementary to a complementary strand of the linear double-stranded nucleic acid template.
5. The method of claim 1 , wherein the second oligonucleotide primer is complementary to a complementary strand of the linear double-stranded nucleic acid template.
6. The method of claim 1 , wherein both the first oligonucleotide primer and the second oligonucleotide primer are complementary to a complementary strand of the linear double-stranded nucleic acid template, and wherein the first oligonucleotide primer and the second oligonucleotide primer are complementary to different strands of the linear double-stranded nucleic acid template.
7. The method of claim 1 , wherein at least one of the first oligonucleotide primer or the second oligonucleotide primer is complementary to at least one of the adaptors.
8. The method of claim 1 , wherein the adaptors contain single strand components of a restriction enzyme recognition sequence corresponding to the same restriction enzyme.
9. The method of claim 1 , wherein the adaptors contain single strand components of a restriction enzyme recognition sequence corresponding to different restriction enzymes.
10. 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 such that under certain conditions they anneal to each other and form the stem of a stem-loop structure, and wherein the stem contains a blunt end.
11. 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 capable of annealing to each other and form the stem of a stem-loop structure, and wherein the outermost part of the stem contains a sticky end.
12. 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 capable of annealing to each other and form the stem of a stem-loop structure, and wherein the outermost part of the stem contains half of a full double-stranded restriction enzyme recognition sequence.
13. The method of claim 1 , wherein the polymerase which generates the extension product of the first primer has strand displacement activity.
14. The method of claim 1 , wherein the polymerase which generates the extension product of the second primer has strand displacement activity.
15. The method of claim 1 , wherein all steps of the method are performed at a temperature of no greater than 70-° C.
16. The method of claim 1 , wherein all steps of the method are performed at a temperature of no greater than 60-° C.
17. The method of claim 1 , wherein all steps of the method are performed at a temperature of no greater than 50-° C.
18. The method of claim 1 , wherein all steps of the method are performed at a temperature of no greater than 40-° C.
19. The method of any 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.