Isothermal strand displacement nucleic acid amplification
View Patent ↗Methods for amplifying target nucleic acid sequences using a nucleic acid polymerase lacking 5′ exonuclease activity and a set of oligonucleotide primers. Preferably, a primer array is used. The primer array contains two sets of primers. One set contains at least two complementary primers. The other set contains at least two sense primers. Using the described methods amplification can be carried out under essentially constant environmental conditions without the requirement for exonuclease activity or restriction endonuclease activity.
1. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of:
providing the following to a sample containing a nucleic acid strand which includes said target sequence:
a nucleic acid polymerase lacking 5′ exonuclease activity;
at least one nucleoside triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate; and
at least two different complementary primers and at least two different sense primers under primer extension conditions,
wherein each said complementary primer contains a nucleotide base sequence which is designed to be complementary to a nucleic acid sequence on said nucleic acid strand located 3′ to said target sequence,
wherein said nucleotide base sequence of each said complementary primer is able to hybridize to a nucleic acid sequence on said nucleic acid strand under said conditions,
wherein each said sense primer contains a nucleotide base sequence which is designed to be analogous to a nucleic acid sequence on said nucleic acid strand located 5′ to said target sequence, and
wherein said nucleotide base sequence of each said sense primer is able to hybridize to a nucleic acid sequence complementary to a nucleic acid sequence on said nucleic acid strand under said conditions; and
amplifying said target sequence at an essentially constant temperature and in the absence of an added restriction endonuclease active on a product of said amplification, wherein said amplifying step is performed in the absence of a complementary or sense primer containing a promoter site for an RNA polymerase.
2. The method of claim 1 , wherein:
said complementary primers which are adjacent to one another are separated at their closest 5′ and 3′ ends by a distance of 1 to 200 nucleotides; and
said sense primers which are adjacent to one another are separated at their closest 5′ and 3′ ends by a distance of 1 to 200 nucleotides.
3. The method of claim 1 , wherein:
said complementary primers which are adjacent to one another are separated at their closest 5′ and 3′ ends by a distance of up to 10 nucleotides; and
said sense primers which are adjacent to one another are separated at their closest 5′ and 3′ ends by a distance of up to 10 nucleotides.
4. The method of claim 1 , wherein at least one of said complementary primers includes a 5′ non-complementary region.
5. The method of claim 1 , wherein said nucleic acid strand is a single strand of DNA and said polymerase is a DNA polymerase.
6. The method of claim 1 , wherein said providing step further includes providing a strand-displacing agent.
7. The method of claim 6 , wherein said strand-displacing agent is a helicase, a RecA protein, or a single-stranded nucleic acid binding protein.
8. The method of claim 1 , wherein said conditions include dimethyl sulfoxide.
9. The method of claim 1 , wherein said essentially constant temperature is at least 43° C.
10. The method of claim 1 , wherein said essentially constant temperature is between 50° C. and 70° C.
11. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of:
providing the following to a sample containing a nucleic acid strand which includes said target sequence:
a nucleic acid polymerase lacking 5′ exonuclease activity;
at least one nucleoside triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate; and
a primer array of at least five different primers comprising at least two or three different complementary primers and at least two or three different sense primers under primer extension conditions,
wherein each said complementary primer contains a nucleotide base sequence which is designed to be complementary to a nucleic acid sequence on said nucleic acid strand located 3′ to said target sequence,
wherein said nucleotide base sequence of each said complementary primer is able to hybridize to a nucleic acid sequence on said nucleic acid strand under said conditions
wherein each said sense primer contains a nucleotide base sequence which is designed to be analogous to a nucleic acid sequence on said nucleic acid strand located 5′ to said target sequence, and
wherein said nucleotide base sequence of each said sense primer is able to hybridize to a nucleic acid sequence complementary to a nucleic acid sequence on said nucleic acid strand under said conditions; and
amplifying said target sequence at an essentially constant temperature, wherein said amplifying step is performed in the absence of a complementary or sense primer containing a promoter site for an RNA polymerase.
12. The method of claim 11 , wherein said amplifying step is carried out in the absence of an added restriction endonuclease active on a product of said amplification.
13. The method of claim 11 , wherein:
said complementary primers which are adjacent to one another are separated at their closest 5′ and 3′ ends by a distance of 1 to 200 nucleotides; and
said sense primers which are adjacent to one another are separated at their closest 5′ and 3′ ends by a distance of 1 to 200 nucleotides.
14. The method of claim 11 , wherein:
said complementary primers which are adjacent to one another are separated at their closest 5′ and 3′ ends by a distance of up to 10 nucleotides; and
said sense primers which are adjacent to one another are separated at their closest 5′ and 3′ ends by a distance of up to 10 nucleotides.
15. The method of claim 11 , wherein at least one of said complementary primers includes a 5′ non-complementary region.
16. The method of claim 11 , wherein said nucleic acid strand is a single strand of DNA and said polymerase is a DNA polymerase.
17. The method of claim 11 , wherein said providing step further includes providing a strand-displacing agent.
18. The method of claim 17 , wherein said strand-displacing agent is a helicase, a RecA protein, or a single-stranded nucleic acid binding protein.
19. The method of claim 11 , wherein said conditions include dimethyl sulfoxide.
20. The method of claim 11 , wherein said essentially constant temperature is at least 43° C.
21. The method of claim 11 , wherein said essentially constant temperature is between 50° C. and 70° C.