METHODS AND KITS FOR THE DETECTION OF NUCLEOTIDE MUTATIONS USING PEPTIDE NUCLEIC ACID AS BOTH PCR CLAMP AND SENSOR PROBE

  • US 20080176226A1
  • Filed: 01/19/2007
  • Published: 07/24/2008
  • Est. Priority Date: 01/19/2007
  • Status: Active Grant
First Claim
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1. 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 the steps of:

  • providing a pair of a first primer and a second primer which 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, the first primer having a sequence identical to that of a first region located upstream of the selected region of the target polynucleotide sequence, the second primer having 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 detectable peptide nucleic acid probe having a sequence that complements fully the sequence of the selected region of the target polynucleotide sequence having no nucleotide variation(s) therein, such that hybridization of the detectable peptide nucleic acid probe to the selected region of the target polynucleotide sequence having no nucleotide variation(s) results in the formation of a duplex having a melting temperature;

    determining the melting temperature of the duplex;

    admixing the detectable peptide nucleic acid probe and the pair of the first primer and the second primer with the nucleic acid sample to form a mixture;

    subjecting the mixture to a PCR process including an extension reaction set to run at a temperature lower than the melting temperature of the duplex by 5 to 20°

    C., such that a mixture of PCR products is obtained; and

    subjecting the mixture of PCR products thus-obtained to a melting analysis to determine melting temperatures of the PCR products, wherein the presence of at least one melting temperature lower than the melting temperature of the duplex is indicative of the nucleotide variation(s) in the selected region of the target polynucleotide sequence contained in the nucleic acid sample.

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