Oligonucleotides containing pyrazolo[3,4-D]pyrimidines for hybridization and mismatch discrimination

A method for primer extension, the method comprising the following steps:

• (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.

A method for determining the nucleotide sequence of a polynucleotide, the method comprising the following steps:

• (a) providing an array of oligonucleotides having different known sequences, wherein one or more purine residues in each of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine;
• (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 the nucleotide sequence of the polynucleotide is determined by examination of the sequences of the oligonucleotides to which the polynucleotide hybridizes.

A method for examining gene expression in a cell, the method comprising the following steps:

• (a) providing a population of polynucleotides representative of the genes expressed in the cell,
• (b) providing an array of oligonucleotides of different sequences, said array comprising a plurality of sites, wherein each site contains a unique oligonucleotide sequence, and wherein one or more purine residues in each of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine;
• (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 detection of the sites on the array at which hybridization occurs is indicative of the pattern of gene expression in the cell.

A method for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, and wherein one or more of the other polynucleotides in the mixture comprise sequences that are related but not identical to the target sequence, the method comprising:

• (a) contacting the mixture of polynucleotides with an oligonucleotide, wherein
• (i) the oligonucleotide has a sequence that is exactly complementary to said target sequence
• (ii) one or more purine residues of the oligonucleotide are substituted by a pyrazolo[3,4-d]pyrimidine,
• (iii) the oligonucleotide forms a stable hybrid with a sequence within said target sequence that is exactly complementary to the oligonucleotide, and
• (iv) the oligonucleotide does not form a stable hybrid with any of the related sequences;
• and
• (b) measuring hybrid formation, whereby hybrid formation is indicative of the presence of said target sequence.