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

A method for detecting the presence of a target sequence in a polynucleotide, the method comprising the following steps:

• (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.

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,
• (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.

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

• (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.

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,
• (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.

A method for identifying a mutation in a target sequence of a gene of interest, the method comprising the following steps:

• (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.