Comparison Of Two Methods Used Ligase Reaction For The Detection Of KRAS, BRAF Gene Mutation

Response to EGFR-targeted therapies in colorectal cancer patients has been convincingly associated with KRAS mutation status. Current mandatory mutation testing for patient selection is limited to the KRAS hotspot codons 12 and 13.Patient carrying mutated KRAS is unlikely to benifit from cetuximab, whereas those carrying wild-type KRAS is likely to benifit from cetuximab.So detection of KRAS mutation in colorectal cancer patients can help the doctor to select patient for anti-EGFR targeted drugs.It can also help doctors choose the most effective treatment method, thus reducing the cost of treatment and side effects.Additionally, some studies have shown that, colorectal cancer patients with mutated BRAF but wild-type KRAS are also unlikely to benifit from cetuximab.So after the detection of KRAS mutation, the detection of BRAF is helpful to exclude patients who are not suitable for cetuximab and other EGFR monoclonal antibody drugs. Therefore, the detection of KRAS mutation firstly then the BRAF is helpful to choose patients who most likely to benefit for anti-EGFR targeted drugs.Therefore, a fast, sensitive, robust method for detection of SNP is in the urgent. There are already many sequence-based or Biological DNA chip for genotyping, however, they either requiring expensive equipment and reagents or requiring cumbersome experimental operation. We now compare two approaches for tracking individual genotypes the gap-ligase chain reaction (Gap-LCR) method and ligase detect reaction (LDR) method firstly,then we selected LDR combination of gel electrophores to detect the KRAS and BRAF mutation. Without expensive laboratory equipment, our method is simple and easy to operate and can detect 5% mutations at least.It can be Carried out in laboratory or hospitals with ordinary conditions. In addition, we used LDR combined Agilent 2100 bio-analyzer to improve the detection flux and one chip can detect 12 samples.We established the positive quality control system in order to get accurate results. We detected KRAS and BRAF V600E mutations in 30 paraffin-embedded cancer tissue samples after the method was established. 4 samples were sequenced directly and the results showed that LDR combination of gel electrophoresis was more sensitive than sequencing. Compared with the MassArray test,more than 50% of the results was consistency of MassArray test. In general, LDR combination of gel electrophoresis results were consistent with the MassArray test. Some tests didn't meet the MassArray result because the inferior sensitivity.But the method is Simple and quick than the MassArray.