Development of a gLCR-based KRAS mutation detection approach and its comparison with other screening methods

A gapped ligase chain reaction (gLCR)-based technique was developed and tested on clinical formalin-fixed, paraffin-embedded (FFPE) tissues from colorectal cancer patients. The technique was designed to detect low-level KRAS codon 12 or 13 mutations or confirming doubtful results gained by less sensitive KRAS screening techniques. The gLCR approach was compared with mutation screening techniques commonly used in routine diagnostics regarding sensitivity and specificity. The herein described monoplex gLCR technique is a useful and powerful tool for detecting low-level KRAS codon 12 and 13 mutations in a vast majority of wild type DNA. The gLCR has the capacity to detect one mutated allele in an excess of at least one million wild type alleles (0.0001 %) and is therefore an ideal technique for confirming doubtful KRAS mutation screening results obtained by other techniques. The variance of the gLCRs signal amplitude was very low and is showing a high reproducibility with constant sensitivity even at higher dilutions. The financial effort and the handling time for this technique are low and comparable to a standard cycle sequencing reaction. Additionally, the gLCR technique is easy extendable for the detection of many other clinical relevant mutation hotspots.