Patent ID: 7491492

Claim:
A method of detecting a mutation in a polynucleotide, wherein the mutation is selected from the group consisting of single nucleotide polymorphisms, short nucleotide tandem repeat mutations, nucleotide deletion mutations, nucleotide insertion mutations, and translocations, said method comprising: producing an elongatable-primer and a counter-primer, wherein the elongatable-primer has (1) a nucleotide sequence A complementary to a portion of said polynucleotide, which portion is 3′ of the mutation to be detected, and (2) a nucleotide sequence B′ added to the 5′ end of sequence A, wherein sequence B′ is complementary to a nucleotide sequence B formed by extending the elongatable-primer hybridized to said portion of said polynucleotide using said polynucleotide as a template, wherein sequence B is immediately 5′ of a sequence or nucleotide complementary to the mutation to be detected, wherein said sequence or nucleotide complementary to the mutation is formed by extending the elongatable-primer hybridized to said portion of said polynucleotide using said polynucleotide as a template, wherein the counter-primer has a Tm less than sequence A and greater than the Tm of sequence B′, wherein the counter-primer hybridizes to the extended elongatable-primer at a site 3′ of said sequence or nucleotide complementary to the mutation to be detected, and wherein the Tm of sequence A is greater than the Tm of sequence B′; producing a target by subjecting the elongatable-primer and the counter-primer to an extension reaction using polymerase and using said polynucleotide as a template at a temperature less than the Tm of sequence A but greater than the Tm of sequence B′; denaturing the target to single-stranded form if the target produced in the step is not single-stranded, such that sequence B′ hybridizes with sequence B in the extended elongatable-primer to form a stem-loop structure; subjecting the stem-loop structure to a hybridization reaction with a probe which is fixed to an electrode, wherein the probe has a nucleic acid sequence at its 3′ end that is complementary to the sequence or nucleotide complementary to the mutation to be detected; subjecting the stem-loop structure and the probe to a ligation reaction to form a ligated target and probe; performing a washing, a denaturing, or a washing and denaturing treatment after the ligation reaction; forming a complementary strand hybridized to the ligated target and probe to form a double-stranded complex; contacting the double-stranded complex with a double strand specific, electrochemically active intercalator; and detecting the electrochemical response of the electrochemically active intercalator with the electrode thereby detecting the mutation.