CRE: a cost effective and rapid approach for PCR-mediated concatenation of and exons KRAS EGFR [version 2; referees: 2 approved]

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 exon 2 and kinase domain exons 18-21 in lung KRAS EGFR 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 o-amplification C of five and exons), followed by concatenation of the PCR product K AS R EGFR 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 L858R and T790M mutations EGFR EGFR in lung cancer cell line and primary tumors. 1 1 1 1 1