Isothermal strand displacement nucleic acid amplification

Abstract

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 endonu- clease activity.

1. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
 contacting a nucleic acid strand containing said target sequence with a nucleic acid polymerase lacking 5′ exonuclease activity, at least one nucleotide triphos- phate selected from adenosine, thymidine, guanosine and cytidine triphosphate, and at least three different complementary primers under primer extension condi- tions at essentially constant temperature, wherein each said 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, and wherein said nucleotide base sequence of each said primer is able to hybridize to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 amplifying said target sequence, wherein said amplifying step is performed in the absence of a complementary or sense primer containing a promoter site for an RNA polymerase.  
 
     
     
       2. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
 contacting a nucleic acid strand containing said target sequence with a nucleic acid polymerase lacking 5′ exonuclease activity, at least one nucleotide triphos- phate selected from adenosine, thymidine, guanosine and cytidine triphosphate, and at least two different complementary primers, under primer extension con- ditions at essentially constant temperature, wherein each said 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, and wherein said nucleotide base sequence of each said primer is able to hybridize to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 amplifying said target sequence in the absence of any 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.  
 
     
     
       3. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
 contacting a nucleic acid strand containing said target sequence with a nucleic acid polymerase, at least one nucleotide triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate, and at least three different complementary primers, under primer extension conditions at essentially constant temperature,  
 wherein each said 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, and wherein said nucleotide base sequence of each said primer is able to hybridize to a nucleic acid sequence on said nucleic acid strand under said conditions, and  
 wherein at least one of said primers is modified to be resistant to nucleolytic degradation by a 5′-exonuclease; and  
 amplifying said target sequence, wherein said amplifying step is performed in the absence of a complementary or sense primer containing a promoter site for an RNA polymerase.  
 
     
     
       4. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of:
 contacting a nucleic acid strand containing said target sequence with a nucleic acid polymerase, at least one nucleotide triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate, and at least two different complementary primers, under primer extension conditions at essentially constant temperature,  
 wherein each said 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, and wherein said nucleotide base sequence of each said primer is able to hybridize to a nucleic acid sequence on said nucleic acid strand under said conditions, and  
 wherein at least one of said primers is modified to be resistant to nucleolytic degradation by a 5′-exonuclease; and  
 amplifying said target sequence in the absence of any 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.  
 
     
     
       5. The method of  claim 1 , wherein at least four said complementary primers are used. 
     
     
       6. The method of  claim 2 , wherein at least four said complementary primers are used. 
     
     
       7. The method of  claim 5 , wherein each of said comple- mentary primers is separated by a distance of 1 to 200 nucleotides. 
     
     
       8. The method of  claim 6 , wherein each of said comple- mentary primers is separated by a distance of 1 to 200 nucleotides. 
     
     
       9. A method for exponentially amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of:
 contacting a nucleic acid strand containing said target sequence with a nucleic acid polymerase lacking 5′ exonuclease activity, at least one nucleotide triphos- phate selected from adenosine, thymidine, guanosine and cytidine triphosphate, and a primer array of at least five different primers comprising two or three different complementary primers and two or three different sense primers, under [oligonucleotide] primer extension con- ditions at essentially constant temperature,  
 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, and 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 of 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 comple- mentary to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 exponentially amplifying said target sequence in the absence of any 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.  
 
     
     
       10. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
 contacting a nucleic acid strand containing said target sequence with a nucleic acid polymerase, at least one-nucleotide triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate, and a primer array of at least five different primers compris- ing two or three different complementary primers and two or three different sense primers, under primer extension conditions at essentially constant temperature,  
 wherein at least one of said primers is modified to be resistant to nucleolytic degradation by a 5′ exonuclease,  
 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, and 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 complementary to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 amplifying said target sequence, wherein said amplifying step is performed in the absence of a complementary or sense primer containing a promoter site for an RNA polymerase.  
 
     
     
       11. A method for exponentially amplifying a specific DNA target sequence preferentially over non-target sequences, comprising the steps of:
 contacting a single stranded DNA containing said target sequence with a DNA polymerase lacking 5′ exonu- clease activity, at least one nucleotide triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate, and a primer array of at least five different primers comprising two or three different complementary primers and two or three different sense primers, under primer extension conditions at essen- tially constant temperature,  
 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, and 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 comple- mentary to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 exponentially amplifying said target sequence in the absence of any 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.  
 
     
     
       12. A method for amplifying a specific DNA target sequence preferentially over non-target sequences, compris- ing the steps of:
 contacting a single stranded DNA containing said target sequence with a DNA polymerase, at least one nucle- otide triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate, and a primer array of at least five different primers comprising two to three different complementary primers and two to three dif- ferent sense primers, under primer extension conditions at essentially constant temperature,  
 wherein at least one of said primers is modified to be resistant to nucleolytic degradation by a 5′ exonuclease,  
 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, and 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 comple- mentary to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 amplifying said target sequence, wherein said amplifying step is performed in the absence of a complementary or sense primer containing a promoter site for an RNA polymerase.  
 
     
     
       13. The method of  claim 9 , wherein at least 4 said sense primers and at least 4 said complementary primers are used. 
     
     
       14. The method of  claim 9 , wherein at least 7 said sense primers and at least 7 said complementary primers are used. 
     
     
       15. The method of  claim 9 , wherein at least 10 said sense primers and at least 10 said complementary primers are used. 
     
     
       16. The method of  claim 9 , wherein each adjacent said sense primer is separated by a distance of 1 to 200 nucleotides, and each adjacent said complementary primer is separated by a distance of 1 to 200 nucleotides. 
     
     
       17. The method of  claim 13 , wherein each adjacent said sense primer is separated by a distance of 1 to 200 nucleotides, and each adjacent said complementary primer is separated by a distance of 1 to 200 nucleotides. 
     
     
       18. The method of  claim 14 , wherein each adjacent said sense primer is separated by a distance of 1 to 200 nucleotides, and each adjacent said complementary primer is separated by a distance of 1 to 200 nucleotides. 
     
     
       19. The method of  claim 15 , wherein each adjacent said sense primer is separated by a distance of 1 to 200 nucleotides, and each adjacent said complementary primer is separated by a distance of 1 to 200 nucleotides. 
     
     
       20. The method of  claim 16 , wherein two adjacent sense primers are separated by a distance of up to 10 nucleotides, and two adjacent complementary primer are separated by a distance of up to 10 nucleotides. 
     
     
       21. A method for exponentially amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of:
 contacting a nucleic acid strand containing said target sequence with a nucleic acid polymerase lacking 5′ exonuclease activity, at least one nucleotide triphos- phate selected from adenosine, thymidine, guanosine and cytidine triphosphate, and at least two different complementary primers and at lease two different sense primers under primer extension conditions at essen- tially constant temperature,  
 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, and 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 comple- mentary to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 exponentially amplifying said target sequence in the absence of any 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.  
 
     
     
       22. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
 contacting a nucleic acid strand containing said target sequence with a nucleic acid polymerase, at least one nucleotide 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 at essentially constant temperature,  
 wherein at least one of said primers is modified to be resistant to nucleolytic degradation by a 5′-exonuclease,  
 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, and 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 comple- mentary to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 amplifying said target sequence in the absence of any 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.  
 
     
     
       23. A method for exponentially amplifying a specific DNA target sequence preferentially over non-target sequences, comprising the steps of:
 contacting a single stranded DNA containing said target sequence with a DNA polymerase lacking 5′ exonu- clease activity, at least one nucleotide triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate, at lease two different comple- mentary primers and at least two different sense prim- ers under primer extension conditions at essentially constant temperature,  
 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, and 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 comple- mentary to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 exponentially amplifying said target sequence in the absence of any restriction endonuclease active on a product of said amplification, wherein said amplifying step is performed in the absence of a primer containing a promoter site for an RNA polymerase.  
 
     
     
       24. A method for amplifying a specific DNA target sequence preferentially over non-target sequences, compris- ing the steps of:
 contacting a single stranded DNA containing said target sequence with a DNA polymerase, at least one nucle- otide triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate, at least two different complementary primers and at least two dif- ferent sense primers under primer extension conditions at essentially constant temperature,  
 wherein at least one of said primers is modified to be resistant to nucleolytic degradation by a 5′ -exonuclease,  
 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, and 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 comple- mentary to a nucleic acid sequence on said nucleic acid strand under said conditions; and  
 amplifying said target sequence in the absence of any 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.  
 
     
     
       25. The method of  claim 23 , wherein said conditions include provision of DMSO. 
     
     
       26. The method of  claim 23 , wherein said essentially constant temperature is at least 43° C. 
     
     
       27. The method of  claim 26 , wherein said essentially constant temperature is between 60° C. and 70° C. 
     
     
       28. The method of  claim 23 , wherein said conditions include provision of a strand-displacing agent. 
     
     
       29. The method of  claim 28 , wherein said strand-displacing agent is selected from the group consisting of a helicase, a RecA protein, and a single-stranded nucleic acid binding protein. 
     
     
       30. The method of  claim 23 , wherein said DNA poly- merase is Bst DNA polymerase I or a modified Taq DNA polymerase. 
     
     
       31. The method of  claim 23 , wherein at least one of the oligonucleotide primers further comprises a 5′ non-complementary region. 
     
     
       32. The method of  claim 23 , wherein at least 4 said complementary primers, and at least 4 said sense primers, are used. 
     
     
       33. The method of  claim 23 , wherein at least 7 said complementary primers, and at least 7 said sense primers, are used. 
     
     
       34. The method of  claim 23 , wherein at least 10 said sense primers, and at least 10 said complementary primers, are used. 
     
     
       35. The method of  claim 10 , wherein at least one said sense primer and one said complementary primer are modified to be resistant to nucleolytic degradation by any 5′-exonuclease present in said contacting step. 
     
     
       36. The method of  claim 12 , wherein at least one said sense primer and one said complementary primer are modi- fied to be resistant to nucleolytic degradation by any 5′-exonuclease present in said contacting step. 
     
     
       37. The method of  claim 22 , wherein at least one said sense primer and one said complementary primer are modi- fied to be resistant to nucleolytic degradation by any 5′-exonuclease present in said contacting step. 
     
     
       38. The method of  claim 24 , wherein at least one said sense primer and one said complementary primer are modi- fied to be resistant to nucleolytic degradation by any 5′-exonuclease present in said contacting step. 
     
     
       39. The method of  claim 10 , wherein said amplifying step is an exponential amplification. 
     
     
       40. The method of  claim 12 , wherein said amplifying step is an exponential amplificatiion. 
     
     
       41. The method of  claim 22 , wherein said amplifying step is an exponential amplification. 
     
     
       42. The method of  claim 24 , wherein said amplifying step is an exponential amplification.