cobas Test Protocol

The cobas^® KRAS Mutation Test kit (cobas test) is a TaqMelt™-based polymerase chain reaction (PCR) assay [20, 21] based on two major processes: (1) manual specimen preparation to obtain genomic DNA from FFPET using the cobas DNA Sample Preparation Kit; and (2) PCR amplification of target DNA. The test is designed to detect 19 mutations in codons 12 and 13 of exon 2 and 61 of exon 3 of the KRAS gene. However, because of the lack of a predicate device for codon 61, we compared the cobas test performance for only codons 12/13.The cobas test has a ≥95 % correct mutation call rate across all specimen types with ~5 % mutant alleles at a DNA input of 0.8–6.3 ng/PCR [20].

Genomic DNA was isolated from at least one slide designated for the cobas test. DNA isolation was performed using the cobas test manufacturer’s instructions to ensure that sufficient DNA was recovered. If the concentration of DNA obtained from a slide was ≥4 ng/μl, testing with the cobas test was performed. If the concentration of the purified sample was <4 ng/μl, DNA isolation was repeated using two slides. Tissue from these two slides was added to one tube and DNA isolation and testing with the cobas test was performed if the concentration of DNA obtained was ≥4 ng/μl. All residual DNA stock from the sample preparation process was quantified and stored at –20 °C for future shipment to a separate clinical laboratory where all samples underwent Sanger sequencing.