Part Of The Denaturing High Performance Liquid Chromatography Detection Of Gastric Cancer Tissues, Plasma And Peritoneal Lavage Fluid Gene Mutation Study

OBJECTIVEDenaturing high - performance liquid chromatography ( DHPLC ) is a relatively new technique developing in recent years. Its main principle to identify DNA sequence variation is as follows: partial - complementary heteroduplexes can form by target sequence with mutation and wild - type sequence after denat-uralization and renaturalization. This kind of heteroduplexes will elute earlier from column than homoduplexes under partial denaturing temperatures. This difference between elution time will help to distinguish variation types. DHPLC has been reported to have the ability to detect the replacement, insertion and deletion of single or multiple base pairs quickly and automatically. It not only is proved to has a higher degree of sensitivity than other available methods but also can detect a wider range of DNA fragments. The PCR product doesn't need special treatment and the price is ten times lower than sequencing at least. Only 8 minutes is required for every single specimen. This technique was applied in our study to detect mutations of relative genes in tumor tissues, blood plasma and washing fluid of abdominal cavity, in order to discuss its application characters in somatic mutation identification of solid tumors and clarify the function of these genes in gastric carcinoma, further more to establish a kind of new time - saving method to screening mutations in gastric carcinoma accurately. Objective: (1) the PCR product amplified from gastric cancer tissue was examined by DHPLC directly without mixing with extra wild type DNA to verify the feasibility. Two main method used in determining the detection temperature were compared to find more optimal temperature. When common Taq polymerase were used in PCR reaction, how to avoid the interference of non -objective peak. (2) Clarify the function of p53, CHK2 and ST7 tumor suppressor gene in gastric cancer by identify their mutation. ( 3 ) Discuss the feasibility of PCR amplification fromserum of gastric cancer patients and establish a new kind of fast method to detect mutations in serum and abdominal washing fluid.MATERIALS AND METHODS1 Collection of specimen: 39 cases of gastric cancer tissue and corresponding adjacent non - tumorous gastric tissue, as well as blood sample and abdominal washing fluid, were obtained by surgical excision from patients in the oncology department of the 1st affiliated hospital of Chinese Medical University from September in 2001 to august in 2002.2 Preparation of DNA: DNA from tissue and abdominal washing fluid was i-solated by a standard proteinase K digestion and phenol - chloroform extraction procedure. DNA from serum was extracted by QIAamp DNA Blood Midi Kit.3 PCR amplification; Routine PCR was conducted except for the denaturing and regeneration process to form heteroduplexes after amplification.4 Selection of detection temperature and DHPLC analysis: Two methods were combined to select optimal temperature and the concentration of B buffer was regulated to ensure the optimal elution time of objective peak. The original DNA extracted from tumor tissue was used for DHPLC analysis directly without mixing with wild type DNA and its detection was conducted parallel to corresponding normal tissue.5 The original PCR products of any tumor sample showing an aberrant DHPLC elution profile and of its corresponding normal tissue sample were purified using a PCR Fragment purification kit (Takara, Dalian) ,then sequenced direct-ly.RESULT1 Application in somatic mutation detection in gastric tumor tissue: Mutations of the p53 gene were investigated in 39 surgical specimens of primary gastric cancer by DHPLC. Altogether, 9 aberrant DHPLC chromatography werefound and all of them were from tumor tissue, while their normal - adjacent counterparts running in parallel showing normal shape. Subsequent sequencing revealed nine sequence variations of 8 mutations and 1 polymorphism. The mutation rate was 21% (8/39). All the 9 aberrant DHPLC chromatography could be detected by either of the two...