Isothermal strand displacement nucleic acid amplification
First Claim
Patent Images
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.
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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.
20 Citations
42 Claims
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1. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
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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. - View Dependent Claims (5, 7)
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2. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
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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. - View Dependent Claims (6, 8)
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3. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
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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, andwherein 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.
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4. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of:
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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, andwherein 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.
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9. A method for exponentially amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of:
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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. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20)
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10. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
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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. - View Dependent Claims (35, 39)
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11. A method for exponentially amplifying a specific DNA target sequence preferentially over non-target sequences, comprising the steps of:
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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.
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12. A method for amplifying a specific DNA target sequence preferentially over non-target sequences, compris- ing the steps of:
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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. - View Dependent Claims (36, 40)
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21. A method for exponentially amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of:
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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.
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22. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, compris- ing the steps of:
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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. - View Dependent Claims (37, 41)
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23. A method for exponentially amplifying a specific DNA target sequence preferentially over non-target sequences, comprising the steps of:
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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. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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24. A method for amplifying a specific DNA target sequence preferentially over non-target sequences, compris- ing the steps of:
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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. - View Dependent Claims (38, 42)
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Specification