Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

<div><p>Establishing the <i>KRAS</i> mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies. Conclusion. Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting.</p></div

Allele-specific primer only amplify sequence-specific templates.

<p>Genomic DNA harbouring G12V or G12V KRAS mutations were used for AsP and Mult-AsP PCR assays, whenever indicated.</p

KRAS mutation analysis relative to NM reference DNA in colorectal carcinoma FFPE tissues.

<p>KRAS mutation analysis relative to NM reference DNA in colorectal carcinoma FFPE tissues.</p

Design of multiplex qPCR for KRAS genotyping.

<p>In a first step was performed PCR to amplify the Non-Mutated reference amplicon KRAS (NM) and simultaneously Multiplex Allele specific (Mult-AsP). When both amplicons were amplified (NM and Mult-AsP), the sample was interpreted as positive and seven allele specific primer (AsP) PCR reactions for the specific identification of mutation were performed. If no amplicon is amplified in the Mult-AsP reaction but amplified in the non-mutated reference reaction, the sample was interpreted as negative or wild type.</p

Sensitivity of the KRAS Multiplex qPCR assay.

<p>Sensitivity of the KRAS Multiplex qPCR assay.</p

ΔCq values of AsP and Mult-AsP qPCR assays.

<p>ΔCq values of AsP and Mult-AsP qPCR assays.</p

AsP and Mult-AsP PCR assays for analysis of the mutational status of <i>KRAS</i> codons 12 and 13 in reference samples.

<p>Genomic DNA of seven reference standards harboring KRAS mutations in codons 12 and 13, was used for AsP and Mult-AsP PCR assay. In all cases the qPCR assays contained the non-mutated reference control reaction (red line). Each reference DNA was amplified in AsP and Multi-AsP PCR (green and blue lines, respectively). In gray curves indicated NTC reaction.</p

KRAS multiplex mutation analysis with colorectal carcinoma FFPE.

<p>Genomic DNA from FFPE tissues were used for AsP and Mult-AsP PCR assay. In all cases the qPCR assays contained the non-mutated reference control reaction (red line). Each DNA was amplified in AsP and Multi-AsP PCR (green and blue lines, respectively). In gray curves indicated NTC reaction.</p