Mutiplex PCR for Detection of Genetic Marker on Cancer in Molecular Epidemiological Study

Objective To design and appreciate a procedure of mutiplex PCR for detection of polymorphism of carcinogenmetabolizing enzymes of lung cancer.Methods Genomic DNA was extracted from the blood of both lung cancer patients and the matched controls by the phenol choroform methods.The three pairs of oligonucleotide primers were synthesized to amplify glutathione S transferase classes mu and theta(GSTM1 and GSTT1) and cytochrome P4501A1(CYP1A1)respectively for detecting the mull genotypes of GSTM1 and GSTT1 and polymorphism of CYP1A1.Results Besides general rules of PCR primer design multiplex PCR primers are required to be stringent specific to the trarget DNA and have not complementary sequence each other.It is also important that the primers should have similar annealing temperature.2.0 mmol/L magnesium and 0.25mmol/L dNTPs were found to be the optimum concentration in this mutiplex PCR.When the annealing temperature is selected the extension time is one of main factors to elevate the yield of products.The ideal time should be long enough to produce the maximum products but should not cause non specific amplification.Conclusion Mutiplex PCR allows to detect more than one genotype simultaneously.It is more efficient and economical compared to the single PCR.However,the concentration of reagents and reaction conditions need to be adjusted repeatedly to guarantee that the results are specific and reliable.