Methods and kits for the detection of nucleotide mutations using peptide nucleic acid as both PCR clamp and sensor probe

A method for determining whether a target polynucleotide sequence contained in a nucleic acid sample has nucleotide variation(s) in a selected region thereof, comprising

• providing a pair of a first primer and a second primer which allows the formation of a PCR product comprising a sequence covering that of the selected region of the target polynucleotide sequence via a PCR process,
• wherein the first primer comprises a sequence based on that of a first region located upstream of the selected region of the target polynucleotide sequence, wherein the second primer comprises a sequence based on that of a second region located downstream of the selected region of the target polynucleotide sequence,
• wherein the 5′-end of the first region is spaced apart from the 5′ end of the sequence of the selected region by 30 nucleotides or more;
• providing a labeled peptide nucleic acid probe comprising a detectable moiety and comprising a sequence that fully complements to the sequence of the selected region of a reference target polynucleotide sequence comprising no nucleotide variation(s) therein, such that hybridization of the labeled peptide nucleic acid probe to the selected region of said reference target polynucleotide sequence results in the formation of a first duplex having a first melting temperature;
• determining the melting temperature of the first duplex;
• admixing the labeled peptide nucleic acid probe and said pair of the first and second primers with a nucleic acid sample to form a mixture;
• subjecting said mixture to a PCR process including an extension reaction set to run at a temperature lower than the melting temperature of the first duplex by 5 to 20° C., such that PCR products are obtained;
• and
• subjecting the PCR products to a melting analysis to determine melting temperatures of the PCR products,
• wherein the presence of at least one melting temperature lower than the first melting temperature of the first duplex is indicative of the nucleotide variation(s) in the selected region of the target polynucleotide sequence contained in the nucleic acid sample.