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

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 one or more purine residues in at least one of the oligonucleotides are substituted by a pyrazolo[3,4-d]pyrimidine, and 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 hybridization of the polynucleotide to oligonucleotides including the wild-type sequence indicates the lack of a mutation, and hybridization of the polynucleotide to oligonucleotides including a mutant sequence indicates the presence of a mutation whose sequence is defined by the particular oligonucleotide to which the polynucleotide is hybridized.

A method for identifying a variant of a target sequence, the method comprising the following steps:

• (a) providing a sample comprising one or more polynucleotides to be tested for the presence of the variant 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 one or more of the other oligonucleotides has a sequence that is perfectly complementary to one or more variant sequences;
• (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;
• wherein hybridization of the polynucleotide to the oligonucleotide having a sequence that is perfectly complementary to the target sequence indicates the absence of a variant, and hybridization of the polynucleotide to an oligonucleotide having a sequence that is perfectly complementary to a particular variant sequence indicates the presence of a variant whose sequence is defined by the oligonucleotide to which the polynucleotide is hybridized.