Kras Gene Detection By Combination Of LDR And Taqman

Cetuxinab and panitumumab are effective drugs of treating colorectal cancer. But not every colorectal cancer patient fit cetuxinab or panitumumab because of their Genetic differences. The KRAS gene is a gene encoding EGFR downstream signaling pathways. For the patients using cetuximab or panitumumab, carrying the wild-type KRAS patients have a better response rate, but the patients with mutant KRAS hardly reach therapeutic effects. KRAS mutations are essentially point mutations, and these mutations focused on the12and13codons of exon2. KRAS has7hotspot mutations, which accounting for90%mutations of KRAS. Therefore, Fast and sensitive detection of these7hotspot mutations is of great significance to colorectal cancerpatient. It not only save on unnecessary expenses for patient, but also promoted achieving personalized medicine for cancer patients.Now, a convenient, fast and high sensitivity methods to detect KRAS is imperative to establish. LDR (Ligase Detect Reaction) is a traditional detection methods, and the main principle is the use of allele-specific primers for genotyping, the results were analyzed by agarose gel electrophoresis. But the drawback is difficult to identify the electrophoresis results, some heterozygotes are not obvious, and the experimental time-comsuming, tedious steps are also its weak points. Taqman is also a commonly method to detect gene expression nowdays, it can record PCR circumstances in real time by fluorophore and quencher of Taqman probe. On this basis, we use Taqman to detect the results of LDR in PCR, to enhance the accuracy of the results, shorten the experimental time and observe the sample genotype intuitively. This method is fast and simple, sensitivity of up to5%or more, and can operate in a normallaboratory environment.Firstly we prepared the positive clone plasmid of KRAS gene, it can be the positive control in experiments and used in the sensitivity experiment. We want to establish the experimental method of LDR combined with Taqman probe, but the7mutations are difficult to detect at the same time, we select the site KRAS13-2to be object of study. For this site, we designed two kinds of Taqman probe, specific probes and common probe. The specific probes correspond to Wild-type and mutant of KRAS13-2and can detect it only; The common probe located upstream of the all mutions and it could detect all of them. The two probes are able to detect KRAS genotype. By optimization experimental conditions and sensitivity experiment, the sensitivity of specific probes and common probe can be achieved by5%, almost the same with LDR, the results are more intuitive and reliable. And then, we improved the structure of P1on the base of LDR, it improved the connection efficiency of LDR and Taqman detection results. But this part of the experiment need to continue to optimize the conditions to obtainbetter experimental progress.