Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

A method for primer extension, comprising the steps of:

• (a) providing a sample containing a target sequence,
• (b) providing one or more oligonucleotide primers complementary to regions of the target sequence,
• (c) providing a polymerizing enzyme and nucleotide substrates, and
• (d) incubating the sample, the oligonucleotide primers, the enzyme and the substrates under conditions favorable for polymerization;
• wherein at least one of the primers comprises a MGB-oligonucleotide conjugate.

A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps:

• (a) providing a polynucleotide comprising a target sequence,
• (b) providing at least two MGB-oligonucleotide conjugates, wherein one of the at least two MGB-oligonucleotide conjugates has a sequence that is perfectly complementary to the target sequence and at least one other of the MGB-oligonucleotide conjugates has a single-nucleotide mismatch with the target sequence;
• (c) separately incubating each of the MGB-oligonucleotide conjugates with the polynucleotide under hybridization conditions;
• and
• (d) determining the hybridization strength between each of the MGB-oligonucleotide conjugates and the polynucleotide.

A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising the following steps:

• (a) providing a MGB-oligonucleotide conjugate of defined sequence,
• (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 MGB-oligonucleotide conjugate and at least one other of the polynucleotides has a target sequence having a single-nucleotide mismatch with the MGB-oligonucleotide conjugate;
• (c) separately incubating each of the polynucleotides with the MGB-oligonucleotide conjugate under hybridization conditions;
• and
• (d) determining the hybridization strength between each of the polynucleotides and the MGB-oligonucleotide conjugate.

A method of ligating two or more oligonucleotides, each of which is hybridized to adjacent sites on a target nucleic acid, comprising the steps of:

• (a) providing a sample containing a target sequence,
• (b) providing at least two oligonucleotides which are complementary to adjacent sites on the target sequence,
• (c) incubating the sample and the oligonucleotides under conditions favorable for ligation, and
• (d) identifying ligated nucleic acids;
• wherein at least one of the oligonucleotides comprises a MGB-oligonucleotide conjugate.

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

• (a) providing an array of oligonucleotide probes of different sequences,
• (b) incubating the polynucleotide and the array under hybridization conditions, and
• (c) determining to which of the oligonucleotide probes in the array the polynucleotide hybridizes;
• wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.

A method for examining gene expression comprising the steps of:

• (a) providing an array of oligonucleotide probes of different sequences,
• (b) incubating a population of polynucleotides with the array under hybridization conditions, and
• (c) determining to which of the oligonucleotide probes in the array the population hybridizes;
• wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.

A method for identifying one or more mutations in a gene of interest comprising the steps of:

• (a) providing an array of oligonucleotide probes of different sequences,
• (b) incubating a polynucleotide sample with the array under hybridization conditions, and
• (c) determining to which of the oligonucleotide probes in the array the polynucleotide hybridizes;
• wherein one or more of the oligonucleotide probes comprises a MGB-oligonucleotide conjugate.