Disclosed herein is a method for determining whether a target polynucleotide sequence contained in a nucleic acid sample has nucleotide variation(s) in a selected region thereof, the steps of which involve the use of a pair of primers that allows the formation of a PCR product having a sequence covering that of the selected region of the target polynucleotide sequence via a PCR process, and a peptide nucleic acid (PNA) that acts as a PCR clamp as well as a sensor probe. Also disclosed herein is a kit for use in determining the presence of nucleotide variation(s) in the target polynucleotide sequence, which includes the pair of primers and the PNA.