Oligonucleotides containing pyrazolo[3,4-d]pyrimidines for hybridization and mismatch discrimination
First Claim
1. A method for distinguishing polynucleotides with related sequences, the method comprising the following steps:
- (a) providing an oligonucleotide having a defined sequence, wherein one or more purine residues of the oligonucleotide are substituted by a pyrazolo[3,4-d]pyrimidine;
(b) providing at least two polynucleotides, each of which comprises a target sequence, wherein one of the polynucleotides has a target sequence that is perfectly complementary to the oligonucleotide and at least one other of the polynucleotides has a related target sequence;
(c) separately incubating each of the polynucleotides with the oligonucleotide under hybridization conditions; and
(d) determining the degree of hybridization between the oligonucleotide and each of the polynucleotides.
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Accused Products
Abstract
Oligonucleotides in which one or more purine residues are substituted by pyrazolo[3,4-d]pyrimidines exhibit improved hybridization properties. Oligonucleotides containing pyrazolo[3,4-d]pyrimidine base analogues have higher melting temperatures than unsubstituted oligonucleotides of identical sequence. Thus, in assays involving hybridization of an oligonucleotide probe to a target polynucleotide sequence, higher signals are obtained. In addition, mismatch discrimination is enhanced when pyrazolo[3,4-d]pyrimidine-containing oligonucleotides are used as hybridization probes, making them useful as probes and primers for hybridization, amplification and sequencing procedures, particularly those in which single- or multiple-nucleotide mismatch discrimination is required.
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Citations
47 Claims
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1. A method for distinguishing polynucleotides with related sequences, the method comprising the following steps:
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(a) providing an oligonucleotide having a defined sequence, wherein one or more purine residues of the oligonucleotide are substituted by a pyrazolo[3,4-d]pyrimidine;
(b) providing at least two polynucleotides, each of which comprises a target sequence, wherein one of the polynucleotides has a target sequence that is perfectly complementary to the oligonucleotide and at least one other of the polynucleotides has a related target sequence;
(c) separately incubating each of the polynucleotides with the oligonucleotide under hybridization conditions; and
(d) determining the degree of hybridization between the oligonucleotide and each of the polynucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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29. A method for detecting the presence of a target sequence in a polynucleotide, the method comprising the following steps:
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(a) providing a polynucleotide which is to be tested for the presence of the target sequence;
(b) providing an oligonucleotide having a sequence that is substantially complementary to the target sequence;
(c) incubating the polynucleotide and the oligonucleotide under hybridization conditions; and
(d) identifying hybridized nucleic acids;
wherein one or more purine residues of the oligonucleotide are substituted by a pyrazolo[3,4-d]pyrimidine. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38)
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39. A method for primer extension, the method comprising the following steps:
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(a) providing a polynucleotide containing a target sequence, (b) providing one or more oligonucleotide primers complementary to the target sequence, (c) providing a polymerizing enzyme and nucleotide substrates, and (d) incubating the polynucleotide, the oligonucleotide primers, the enzyme and the substrates under conditions favorable for polymerization;
wherein one or more purine residues of the one or more oligonucleotide primers are substituted by a pyrazolo[3,4-d]pyrimidine. - View Dependent Claims (40, 41, 42)
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43. A method for determining the nucleotide sequence of a polynucleotide, the method comprising the following steps:
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(a) providing an array of oligonucleotides having different known sequences, (b) incubating the polynucleotide with the array under hybridization conditions, and (c) determining to which of the oligonucleotides in the array the polynucleotide hybridizes;
wherein one or more purine residues in each of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine.
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44. A method for determining the nucleotide sequence of a target sequence in a polynucleotide, the method comprising the following steps:
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(a) providing a polynucleotide comprising the target sequence;
(b) providing at least two oligonucleotides of known sequence wherein one or more purine residues of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine, and wherein one of the at least two oligonucleotides has a sequence that is perfectly complementary to the target sequence and at least one other of the oligonucleotides has a related target sequence;
(c) separately incubating each of the oligonucleotides with the polynucleotide under hybridization conditions; and
(d) determining the degree of hybridization between each of the oligonucleotides and the polynucleotide. - View Dependent Claims (45)
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46. A method for examining gene expression in a cell, the method comprising the following steps:
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(a) providing a population of polynucleotides representative of the genes expressed in the cell, (b) providing an array of oligonucleotides of different sequences, (c) incubating the population of polynucleotides with the array under hybridization conditions, and (d) determining which of the oligonucleotides in the array become hybridized to polynucleotides;
wherein one or more purine residues in each of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine.
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47. A method for identifying a mutation in a target sequence of a gene of interest, the method comprising the following steps:
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(a) providing a polynucleotide that comprises the target sequence, (b) providing an array of oligonucleotides of different sequences, wherein the different sequences include the wild-type target sequence and different mutant target sequences, (c) incubating the polynucleotide with the array under hybridization conditions, and (d) determining which of the oligonucleotides in the array become hybridized to the polynucleotide;
wherein one or more purine residues in each of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine.
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Specification