Nucleic acid sequence amplification methods
First Claim
Patent Images
1. A method of synthesizing multiple copies of a target nucleic acid sequence which consists essentially of:
- (a) treating a nucleic acid which comprises an RNA target sequence with a first oligonucleotide which comprises a first primer which has a hybridizing sequence sufficiently complementary to a 3'"'"'-terminal portion of the target sequence to hybridize therewith and which optionally has a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated;
(b) extending the primer in an extension reaction using the target as a template to give a DNA primer extension product complementary to the RNA target;
(c) separating the DNA primer extension product from the RNA target using an enzyme which selectively degrades the RNA target;
(d) treating the DNA primer extension product with a second oligonucleotide which comprises a primer or a splice template and which has a hybridizing sequence sufficiently complementary to the 3'"'"'-terminal portion of the target sequence to hybridize therewith, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated, provided that if the first oligonucleotide does not have a promoter, then the second oligonucleotide is a splice template which has a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase;
(e) extending the 3'"'"'-terminus of either the second oligonucleotide or the first primer extension product, or both, in a DNA extension reaction to produce a template for an RNA polymerase; and
(f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter sequence;
wherein said method is conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNAse H activity is used in the method.
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Abstract
Methods of synthesizing multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH are provided in which multiple RNA copies of the target sequence autocatalytically generate additional copies. These methods are useful for generating copies of a nucleic acid target sequence for purposes which include assays to quantitate specific nucleic acid sequences in clinical, environmental, forensic and similar samples, cloning and generating probes.
977 Citations
49 Claims
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1. A method of synthesizing multiple copies of a target nucleic acid sequence which consists essentially of:
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(a) treating a nucleic acid which comprises an RNA target sequence with a first oligonucleotide which comprises a first primer which has a hybridizing sequence sufficiently complementary to a 3'"'"'-terminal portion of the target sequence to hybridize therewith and which optionally has a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated; (b) extending the primer in an extension reaction using the target as a template to give a DNA primer extension product complementary to the RNA target; (c) separating the DNA primer extension product from the RNA target using an enzyme which selectively degrades the RNA target; (d) treating the DNA primer extension product with a second oligonucleotide which comprises a primer or a splice template and which has a hybridizing sequence sufficiently complementary to the 3'"'"'-terminal portion of the target sequence to hybridize therewith, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated, provided that if the first oligonucleotide does not have a promoter, then the second oligonucleotide is a splice template which has a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase; (e) extending the 3'"'"'-terminus of either the second oligonucleotide or the first primer extension product, or both, in a DNA extension reaction to produce a template for an RNA polymerase; and (f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter sequence;
wherein said method is conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNAse H activity is used in the method. - View Dependent Claims (11)
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2. A method of synthesizing multiple copies of a target nucleic acid sequence which consists essentially of:
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(a) treating a target nucleic acid which comprises an RNA target sequence with a first oligonucleotide which comprises a first primer which has a hybridizing sequence sufficiently complementary to a 3'"'"'-terminal portion of the target sequence to hybridize therewith and a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated; (b) extending the primer in an extension reaction using the target as a template to give a first DNA primer extension product complementary to the RNA target; (c) separating the first DNA primer extension product from the RNA target using an enzyme which selectively degrades the RNA target; (d) treating the first DNA primer extension product with a second oligonucleotide which comprises a second primer which has a hybridizing sequence sufficiently complementary to the 3'"'"'-terminal portion of the target sequence to hybridize therewith, under conditions whereby an oligonucleotide/target sequence is formed and DNA synthesis may be initiated; (e) extending the 3'"'"'-terminus of the second primer in a DNA extension reaction to give a second DNA primer extension product, thereby producing a template for an RNA polymerase; and (f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter sequence;
wherein said method is conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNase H activity is used in the method. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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3. A method of synthesizing multiple copies of a target nucleic acid sequence which consists essentially of:
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(a) treating a nucleic acid which comprises an RNA target sequence with a first oligonucleotide which comprises a first primer which has a hybridizing sequence sufficiently complementary to a 3'"'"'-terminal portion of the target sequence to complex therewith, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated; (b) extending the 3'"'"' terminus of the first primer in an extension reaction using the target as a template to give a DNA primer extension product complementary to the RNA target; (c) separating the DNA primer extension product from the RNA target using an enzyme which selectively degrades the RNA target; (d) treating the DNA primer extension product with a second oligonucleotide which comprises a splice template which has a hybridizing sequence sufficiently complementary to the 3'"'"' terminus of the primer extension product to hybridize therewith and a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated; (e) extending the 3'"'"'-terminus of the primer extension product to add thereto a sequence complementary to the promoter, thereby producing a template for an RNA polymerase; and (f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter;
wherein said method is conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising. RNase H activity is used in the method. - View Dependent Claims (24, 25, 26, 27, 28, 29, 48)
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4. A method of synthesizing multiple copies of a target sequence which consists essentially of:
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(a) treating a single stranded target nucleic acid which comprises a DNA target sequence having a defined 3'"'"' terminus with a first oligonucleotide which comprises a splice template which has a hybridizing sequence sufficiently complementary to a 3'"'"' terminal portion of the target sequence to hybridize therewith and a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated; (b) extending the 3'"'"' terminus of the target to add a sequence complementary to the promoter, thereby producing a template for an RNA polymerase; and (c) using the template of step (b) to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter;
wherein said method is conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNase H activity is used in the method. - View Dependent Claims (30, 31, 32, 33, 34)
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5. A method of synthesizing multiple copies of a target nucleic acid sequence which consists essentially of:
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(a) treating a single stranded target nucleic acid which comprises a DNA target sequence with a first oligonucleotide which comprises a first primer which has a hybridizing sequence sufficiently complementary to a 3'"'"'-terminal portion of the target sequence to hybridize therewith and a sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated; (b) extending the primer in a DNA extension reaction using the target as a template to give a first DNA primer extension product complementary to the DNA target; (c) separating the first primer extension product from the target; (d) treating the first primer extension product with a second oligonucleotide which comprises a second primer which has a hybridizing sequence sufficiently complementary to the 3'"'"'-terminal portion of the target sequence to hybridize therewith, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated; (e) extending the 3'"'"' terminus of the second primer in a DNA extension reaction to give a second DNA primer extension product, thereby producing a template for a RNA polymerase; (f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter sequence; (g) treating an RNA copy from step (f) with the second primer under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated; (h) extending the 3'"'"'-terminus of the second primer in a DNA extension reaction to give a second DNA primer extension product complementary to the RNA copy; (i) separating the DNA primer extension product from the RNA copy using an enzyme which selective degrades the RNA copy; (j) treating the second primer extension product with the first primer, under conditions whereby an oligonucleotide/target hybrid is formed and DNA synthesis may be initiated; (k) extending the 3'"'"'-terminus of the first primer in a DNA extension reaction to give a first DNA primer extension product and the 3'"'"'-terminus of the second primer extension product, thereby producing a template for an RNA polymerase; and (l) using the template of step (k) to produce multiple copies of the target sequence using an RNA polymerase which recognizes the promoter;
wherein said steps (d) through (l) are conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNase H activity is used in the method. - View Dependent Claims (35, 36, 37)
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6. A method of synthesizing multiple copies of a target nucleic acid sequence which consists essentially of:
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(a) treating a single stranded target nucleic acid which comprises a DNA target sequence with a first oligonucleotide which comprises a primer which has a hybridizing sequence sufficiently complementary to a 3'"'"'-terminal portion of the target sequence to hybridize therewith under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated; (b) extending the 3'"'"'-terminus of the primer in an extension reaction using the target as a template to give a DNA primer extension product complementary to the target; (c) separating the primer extension product from the target; (d) treating the primer extension product with a second oligonucleotide which comprises a splice template which has a hybridizing sequence sufficiently complementary to the 3'"'"' terminal portion of the target sequence to hybridize therewith and a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence hybrid is formed and DNA synthesis may be initiated; (e) extending the 3'"'"'-terminus of the primer extension product to add thereto a sequence complementary to the promoter, thereby producing a template for the RNA polymerase; and (f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA polymerase which recognizes the promoter;
wherein said steps (d) through (f) are conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNase H activity is used in the method. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 49)
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7. A method for synthesizing multiple copies of a target nucleic acid sequence under conditions of constant temperature which consists essentially of:
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(a) combining (1) a target nucleic acid which comprises a single-stranded RNA target sequence; (2) a first oligonucleotide which comprises a primer which has a hybridizing sequence sufficiently complementary to a 3'"'"' terminal portion of the RNA target sequence to hybridize therewith and which optionally has a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase; (3) a second oligonucleotide which comprises a primer or a splice template and which has a hybridizing sequence sufficiently complementary to the 3'"'"'-terminal portion of a complement to the RNA target sequence to hybridize therewith, provided that if the first oligonucleotide does not have a promoter, then the second oligonucleotide is a splice template which has a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase; (4) a DNA polymerase; (5) an enzyme which selectively degrades the RNA strand of an RNA;
DNA duplex; and(6) an RNA polymerase which recognizes the promoter; and (b) incubating the mixture of step (a) under DNA priming and nucleic acid synthesizing conditions, which include constant temperature; and
wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNase H activity is used in the method.
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8. A method for synthesizing multiple copies of a target nucleic acid sequence under conditions of constant temperature which consists essentially of:
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(a) combining; (1) a target nucleic acid which comprises an RNA target sequence; (2) a primer and a splice template of opposite sense wherein one has a hybridizing sequence sufficiently complementary to a 3'"'"'-terminal portion of the target sequence of the RNA target to hybridize therewith and the other has a hybridizing sequence sufficiently complementary to the 3'"'"' terminal portion of the target sequence of its complement to hybridize therewith and wherein the splice template has a sequence 5'"'"' to the hybridizing sequence which includes a promoter; (3) a DNA polymerase; (4) an enzyme which selectively degrades the RNA strand of an RNA;
DNA duplex; and(5) an RNA polymerase which recognizes the promoter of the splice template; and (b) incubating the mixture of step (a) under DNA priming and nucleic acid synthesizing conditions which include constant temperature; and
wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNase H activity is used in the method.
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9. A method for synthesizing multiple copies of a target nucleic acid sequence which consists essentially of:
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(a) combining; (1) a nucleic acid which comprises a single stranded DNA target sequence; (2) a first primer which has a hybridizing sequence sufficiently complementary to a 3'"'"'-terminal portion of the target sequence to hybridize therewith and a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase; (3) a DNA polymerase; (b) incubating the mixture of step (a) under DNA priming and synthesizing conditions whereby a primer extension product complementary to the target sequence is synthesized using the target sequence as a template; (c) treating the reaction mixture of step (b) to cause separation of DNA duplexes; (d) adding to the reaction mixture of step (c); (1) a second primer which has a hybridizing sequence sufficiently complementary to the 3'"'"'-terminal portion of the target sequence of the primer extension product to hybridize therewith; (2) a DNA polymerase (3) an enzyme which selectively degrades the RNA strand of an RNA;
DNA complex; and(4) an RNA polymerase which recognizes the promoter; and (e) incubating the mixture of step (d) under DNA priming and nucleic acid synthesizing conditions which include constant temperature; and
wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNase H activity is used in the method.
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10. A method for synthesizing multiple copies of a target nucleic acid sequence under conditions of constant temperature which consists essentially of:
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(a) combining; (1) a nucleic acid which comprises a target sequence; (2) a first oligonucleotide which comprises first primer which has a hybridizing sequence sufficiently complementary to a 3'"'"' terminal portion of the target sequence to hybridize thereto and which optionally has a sequence 5'"'"' to the hybridizing sequence which includes a promoter for an RNA polymerase; (3) a second oligonucleotide which comprises a second primer or a splice template and which has a hybridizing sequence sufficiently complementary to the 3'"'"' terminal portion of the target sequence to hybridize thereto, provided that if the first oligonucleotide does not have a promoter, then the second oligonucleotide is a splice template, which has a sequence 5'"'"' to the hybridizing sequence which includes a promoter; (4) a DNA polymerase (5) an enzyme which selectively degrades the RNA strand of an RNA;
DNA duplex; and(6) an RNA polymerase; and (b) incubating the mixture of step (a) under DNA priming and nucleic acid synthesizing conditions, which include constant temperature; and
wherein a reverse transcriptase comprising RNase H activity is used in the method, and no other enzyme comprising RNase H activity is used in the method. - View Dependent Claims (45, 46, 47)
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Specification