The Study Of Single Nucleotide Polymorphisms And Expression Of RRM1 And Mdr1 Gene

Among cancers, lung cancer is the leading cause of death.It is responsible for 28% of cancer mortality and causes more deaths than colorectal cancer, breast cancer,and prostate cancer combined. Lacking of the methods of early-diagnose and treatment ,three quarters of the preliminary diagnosis patients have no chance of surgery. No chemotherapy regimen, including the widely used combination of gemcitabine /cisplatin ,confers significantly improved survival over any other in metastatic non small cell lung cancer; studies indicate some polymorphisms of target-gene and expression effect the response of chemotherapy. However, the selection of patients according to key genetic characteristics can help to tailor chemotherapy.Real-time quantitative PCR and Genechip (oligonucleotide microarray) are technology developed in recent years which provides a powerful high throughout data mining and analyzing platform for the research and discovery in the fields of modern biomedicine, and is increasingly widely used in the detection of gene in the pathogens responsible for malignant diseases.In the first part of this paper, we established a simple and specific assay for the genetyping of RRM1 (-)37A/C. PCR reactions for genotyping (-)37A/C by using allele-specific primers were conducted in separate tubes. An additional nucleotide mismatch at the third position from the 3' end of each allele-specific primer was used to abrogate nonspecific PCR amplification. The fluorescence emitted by SYBR Green I was monitored to detect formation of specific PCR products. PCR growth curves exceeding the threshold cycle were considered positive. Fluorescence melt-curve analysis was used to corroborate results from PCR growth curves. The results showed that 0.25μmol/L primers was optimal for this quantitative PCR assay. This method is thorough with good accuracy and specificity. The correlation between Ct values and starting concentration of plasmid RRM1 is linear and accurate quantitative can be achieved with samples detected within 10~1-10~6 copies/ul. The reproducibility of the assay was also investigated, it showed the coefficient of variation of intra-assay and inter-assay was less than 5 %. The frequency of the allele gene A is 31.3% in Chinese people. SYBR Green I real time PCR appeared to be a rapid accurate and specific...