CRE: a cost effective and rapid approach for PCR-mediated concatenation of KRAS and EGFR exons

Abstract

Molecular diagnostics has changed the way lung cancer patients are treated worldwide. Of several different testing methods available, PCR followed by directed sequencing and amplification refractory mutation system (ARMS) are the two most commonly used diagnostic methods worldwide to detect mutations at KRAS exon 2 and EGFR kinase domain exons 18-21 in lung cancer. Compared to ARMS, the PCR followed by directed sequencing approach is relatively inexpensive but more cumbersome to perform. Moreover, with a limiting amount of genomic DNA from clinical formalin-fixed, paraffin-embedded (FFPE) specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions often get challenging. Here, we report a cost-effective single multiplex-PCR based method, CRE (for Co-amplification of five K RAS and E GFR exons), followed by concatenation of the PCR product as a single linear fragment for direct sequencing. CRE is a robust protocol that can be adapted for routine use in clinical diagnostics with reduced variability, cost and turnaround time requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples. As a proof of principle, CRE is able to detect the activating EGFR L858R and T790M EGFR mutations in lung cancer cell line and primary tumors.

CRE: a cost effective and rapid approach for PCR-mediated concatenation of KRAS and EGFR exons

“…Concatenated KRAS and EGFR (CRE) product of ~915 bp amplified with OAD 176 and OAD 152 using multiplex PCR product as template derived from NCI-H1975 genomic DNA (shown in Panel B , Lane 2); derived from fresh frozen primary tumor genomic DNA (shown in Panel C , Lane 2); using tumor genomic DNA extracted from FFPE block (shown in Panel D, Lane 2) ( Ramteke et al , 2015a). …”

“…Panel A displays 15 nucleotide junction region flanked by KRAS exon 2 and EGFR exon 18 sequence; Panel B displays 15 nucleotide junction region flanked by EGFR exons 18 and 19; Panel C displays 15 nucleotide junction region flanked by EGFR exons 19 and 20; and displays 15 nucleotide junction region flanked by EGFR exons 20 and 21 is shown in Panel D ( Ramteke et al , 2015b). …”

“…Panel A: The arrow indicates expected location of the wild-type and T790M mutant allele peak. Panel B: The arrow indicates expected location of the wild-type and L858R mutant allele peak ( Ramteke et al , 2015c). …”

“…The arrow indicates expected location of the wild-type and L858R mutant allele peak ( Ramteke et al , 2015d). …”