Method for producing a polynucleotide for use in single primer amplification
First Claim
1. A method for forming, from an extender probe, which maybe extended or modified, and a single stranded target polynucleotide sequence, a polynucleotide, said polynucleotide being complementary to an extended extender probe and having a sequence identical to said target polynucleotide sequence attached at its 3'"'"'-end to a polynucleotide sequence complementary to a polynucleotide sequence at the 5'"'"' end of said target polynucleotide sequence, wherein during said method said extender probe is modified to enhance the efficiency of said forming of said polynucleotide by reducing the priming effectiveness of said extender probe, said method comprising:
- (a) hybridizing to a sequence S1 at the 3'"'"'-end of said single stranded target polynucleotide sequence the 3'"'"'-end of said extender probe wherein said extender probe contains a sequence substantially identical to a sequence S2 at the 5'"'"'-end of said single stranded target polynucleotide sequence and wherein said S1 and said S2 are non-complementary and separated from one another by at least 10 nucleotides,(b) extending, by means of a polydeoxynucleotide polymerase and deoxynucleoside triphosphates, said extender probe along said single stranded target polynucleotide sequence to produce an extended extender probe,(c) extending or degrading by means of an enzyme said 3'"'"'-end of said extender probe not hybridized to said single stranded target polynucleotide sequence to produce a modified extender probe,(d) hybridizing a primer to the 3'"'"'-end of the extended extender probe, said primer having said sequence S2 at its 3'"'"'-end, and(e) extending, by means of a polydeoxynucleotide polymerase and deoxynucleoside triphosphates, said primer along said extended extender probe, thereby forming said polynucleotide having a sequence identical to said target polynucleotide sequence attached at its 3'"'"'-end to a polynucleotide sequence complementary to a polynucleotide sequence at the 5'"'"' end of said target polynucleotide sequence.
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Abstract
A method is disclosed for extending an extender probe to produce a single stranded polydeoxynucleotide that is free of unreacted extender probe and has two segments that are non-contiguous and complementary with each other. The method comprises the steps of (1) providing in combination (a) a polynucleotide having two non-contiguous, non-complementary nucleotide sequences S1 and S2 wherein S2 is 5'"'"' of S1 and is at least ten deoxynucleotides long, (b) an extender probe comprised of two deoxynucleotide sequences, wherein the sequence at the 3'"'"'-end of the extender probe (EP1) is hybridizable with S1 and the other of the deoxynucleotide sequences (EP2) is substantially identical to S2 and (c) means for modifying the 3'"'"'-end of extender probe that does not hybridize with the polynucleotide and (2) extending the extender probe along the polynucleotide wherein extender probe not hybridized to the polynucleotide becomes modified at its 3'"'"'-end.
79 Citations
46 Claims
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1. A method for forming, from an extender probe, which maybe extended or modified, and a single stranded target polynucleotide sequence, a polynucleotide, said polynucleotide being complementary to an extended extender probe and having a sequence identical to said target polynucleotide sequence attached at its 3'"'"'-end to a polynucleotide sequence complementary to a polynucleotide sequence at the 5'"'"' end of said target polynucleotide sequence, wherein during said method said extender probe is modified to enhance the efficiency of said forming of said polynucleotide by reducing the priming effectiveness of said extender probe, said method comprising:
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(a) hybridizing to a sequence S1 at the 3'"'"'-end of said single stranded target polynucleotide sequence the 3'"'"'-end of said extender probe wherein said extender probe contains a sequence substantially identical to a sequence S2 at the 5'"'"'-end of said single stranded target polynucleotide sequence and wherein said S1 and said S2 are non-complementary and separated from one another by at least 10 nucleotides, (b) extending, by means of a polydeoxynucleotide polymerase and deoxynucleoside triphosphates, said extender probe along said single stranded target polynucleotide sequence to produce an extended extender probe, (c) extending or degrading by means of an enzyme said 3'"'"'-end of said extender probe not hybridized to said single stranded target polynucleotide sequence to produce a modified extender probe, (d) hybridizing a primer to the 3'"'"'-end of the extended extender probe, said primer having said sequence S2 at its 3'"'"'-end, and (e) extending, by means of a polydeoxynucleotide polymerase and deoxynucleoside triphosphates, said primer along said extended extender probe, thereby forming said polynucleotide having a sequence identical to said target polynucleotide sequence attached at its 3'"'"'-end to a polynucleotide sequence complementary to a polynucleotide sequence at the 5'"'"' end of said target polynucleotide sequence.
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2. A method for producing from an extender probe a polydeoxynucleotide, said polydeoxynucleotide being an extended extender probe and having two segments that are non-contiguous and complementary with each other, wherein during said method said extender probe is modified to reduce the priming effectiveness thereof in an amplification of said polydeoxynucleotide, said method comprising:
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providing in combination (a) a polynucleotide having two non-contiguous, non-complementary nucleotide sequences S1 and S2 wherein S2 is 5'"'"' of S1 and is at least ten nucleotides long wherein said S1 and S2 are separated by at least 10 nucleotides, (b) an extender probe comprised of two deoxynucleotide sequences, wherein the sequence at the 3'"'"'-end of said extender probe, EP1, hybridizes with S1 and the other of said deoxynucleotide sequences, EP2, is homologous to S2, and (c) an enzyme for chemically modifying the 3'"'"'-end of said extender probe that does not hybridize with said polynucleotide, and extending, by means of a polydeoxynucleotide polymerase and polydeoxynucleoside triphosphates, said extender probe along said polynucleotide to produce extended extender probe which is said polydeoxynucleotide having two segments that are non-contiguous and complementary with each other, wherein the 3'"'"'-end of said extender probe not hybridized with said polynucleotide is extended or degraded by means of said enzyme, thereby producing modified extender probe. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for replicating a target polynucleotide sequence, said target polynucleotide sequence having two non-contiguous, non-complementary nucleotide sequences S1 and S2 each at least 10 nucleotides long separated from one another by at least 10 nucleotides, wherein S2 is 5'"'"' of S1 and wherein during said method said extender probe is extended or degraded to reduce the priming effectiveness thereof in further replication of said target polynucleotide sequence, said method comprising:
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providing in combination, either concomitantly or wholly or partially sequentially, (1) said target polynucleotide sequence, (2) an extender probe, which may be extended_or modified, having two deoxynucleotide sequences wherein the sequence at the 3'"'"'-end of said extender probe, EP1, hybridizes with S1 and the other of said deoxynucleotide sequences, EP2, is homologous to S2, (3) an enzyme for extending or degrading the 3'"'"'-end of said extender probe not hybridized with said target polynucleotide sequence, (4) a polydeoxynucleotide primer comprised of sequence S2 at its 3'"'"'-end where said polydeoxynucleotide primer may be provided directly or generated in situ, (5) DNA polymerase and (6) deoxynucleoside triphosphates under conditions wherein (A) some of said extender probe becomes hybridized with and extended along said target polynucleotide sequence to form a duplex comprising extended extender probe, (B) extender probe not hybridized to said target nucleotide sequence is extended or degraded at its 3'"'"'-end by said enzyme, (C) said extended extender probe is dissociated from said duplex, (D) said polydeoxynucleotide primer hybridizes with and is extended along said extended extender probe to form a duplex comprising extended polydeoxynucleotide primer, (E) said extended polydeoxynucleotide primer is dissociated from said duplex, and (F) said polydeoxynucleotide primer hybridizes with and is extended along said extended polydeoxynucleotide primer to form a duplex comprising extended polydeoxynucleotide primer and steps (E) and (F) are repeated, wherein said extended polydeoxynucleotide primer is a replication of said target polynucleotide sequence. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A kit comprising in packaged combination:
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an extender probe having at its 3'"'"'-end a sequence (EP1) that hybridizes with a first sequence in a target polynucleotide sequence and having a sequence (EP2) that is substantially identical to a second sequence of said target polynucleotide sequence, wherein in said target polynucleotide sequence said second sequence is 5'"'"' and non-contiguous with said first sequence, a nucleotide sequence (NS) having a portion that hybridizes with EP1 wherein said NS may be a separate molecule or part of said extender probe, and a polydeoxynucleotide primer that hybridizes with a sequence that is complementary with said second sequence. - View Dependent Claims (34, 35, 36, 37, 38)
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39. A kit comprising in packaged combination:
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an extender probe having at its 3'"'"'-end a sequence that hybridizes with a first sequence (EP1) in a target polynucleotide sequence and having a sequence that is substantially identical to a second sequence (EP2) of said target polynucleotide sequence, wherein in said target polynucleotide sequence said second sequence is 5'"'"' and non-contiguous with said first sequence, an enzyme that degrades said sequence that hybridizes with said EP1 when said sequence is not hybridized to said EP1, and a polydeoxynucleotide primer that hybridizes with a sequence that is complementary with said second sequence. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46)
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Specification