Method for the detection of egfr mutations in blood samples

Abstract

The present invention refers to the detection of EGFR mutations in a blood (serum/plasma) sample from a subject. The method comprises: (i) obtaining the DNA from said sample; (ii) amplifying the nucleic acid sequence corresponding to a specific region of the EGFR gene by means of PCR using a Protein-Nucleic Acid probe; and (iii) detecting said mutation.

1 . A method for the detection of mutations at the EGFR gene in a serum or plasma sample from a subject, said method comprising:
 (i) obtaining DNA from said sample;   (ii) amplifying a nucleic acid sequence corresponding to a specific target region of the EGFR gene comprising a mutation to be detected by means of PCR using primers capable of amplifying said target region of the EGFR gene and a Protein-Nucleic Acid probe wherein said Protein-Nucleic Acid probe is capable of specifically recognizing and hybridizing with the EGFR wild-type sequence within said specific region of the EGFR gene thereby inhibiting the amplification of the wild-type sequence and producing PCR products from the gene comprising the mutation to be detected; and   (iii) detecting the PCR products obtained in (ii)   whereby the detection of the PCR product indicates that there is a mutation in the EGFR gene.   
     
     
         2 . The method according to  claim 1 , wherein the mutation at nucleic acid sequence corresponding to a specific region of the EGFR gene to be detected comprises a mutation is-selected from the group consisting of: ELREA deletions at the exon 19, an L858R mutation at the exon 21, and a T790M mutation at the exon 20. 
     
     
         3 . The method according to  claim 1 , wherein the detecting is carried out by means of nucleic acid sequencing. 
     
     
         4 . The method according to  claim 1 , wherein the amplification is carried out using a primer labeled with one detectable moiety and wherein the detecting step is carried out by means of capillary electrophoresis. 
     
     
         5 . The method according to  claim 4 , wherein said the primer labeled with one detectable moiety is an oligonucleotide primer which is fluorescently labelled at its 5′ end with a fluorescent dye. 
     
     
         6 . The method of  claim 5 , wherein said fluorescent dye is selected from the group consisting of 6-FAM, HEX and NED dyes. 
     
     
         7 . The method of  claim 1 , wherein the detection of the PCR product is carried out using a 5′ nuclease activity assay.