Detection Of EphB2 Mutation In Human Gastric Cancer By DHPLC

IntroductionGastric cancer is one of the most important malignant cancers in China which causes the highest morbility and mortality . The procession of gastric cancer tumourigenesis and progression is multigenetic and multistage including onco-gene activation and tumor suppressor genes inactivation. In this complicated procession , gene mutation is an important mechanism . Eph ( erythropoietin — producing hepatoma amplified sequence) family is the largest receptor tyrosine protein kinase family in human genome including 14 types of receptor tyrosine protein kinase and has many biological functions in human body . Some recent studies show that EphB2 gene is involved in the tumourigenesis and progression procession of gastric cancer.DHPLC ( Denaturing High Performance Liquid Chromatography) is a new high - flux technique to detect gene mutation . It has many virtues: 1) it can detect 1 sample in 8. 8 minutes;2) it has automatic mode of operation;3)it can e-valuate the size of DNA fragment accurately;4) the detection rate reaches up to 95% -100%;5)it' s economical and convenient ,PCR product can be detected without purification.We detect EphB2 gene mutation in human gastric cancer to find the relationship between EphB2 gene and gastric cancer and its function mode in the procession of gastric cancer tumourigenesis and progression.Materials and Methods1. Materials1. 1 Specimen;40 samples of gastric tumors were obtained from the Department of Surgical Oncology of the No. 1 Affiliated Hospital of China Medical University during surgery, including 24 males and 16 females.1. 2 main laboratory apparatus and reagent: DHPLC( TRANSGENE LTD. ,USA) ., PCR(TAKARA LTD. , JAPAN) , tissue homogenizer (HEIDOLPH, Germany) , GEL ANALYSATOR( BIO - RAD CO. ,USA);PCR KIT and gel reclaimation kit (TAKARA LTD. JAPAN) , DHPLC( TRANSGENE LTD. ,USA)2. Methods2. 1 Specimen collection: Specimen is placed in aseptic consple after being cut off from human body. Then cut Specimen open along the opposite side of cancer, wash gastric contents with SPSS . Cut off about lxl x 1 cm3 cancer tissue from the borderline of cancer, envelop it in tinfoil paper and stored at -80^.2. 2 Genome DNA Extraction: put cancer tissue into digestive juice and extract genome DNA, dissolve it in TE.2. 3 PCR amplification: use proper primers and genome DNA as template to amplify the DNA fragment contented 17 extrons DNA sequence of EphB2 gene by PCR, respectively.2.4 DHPLC detection;PCR products are denaturalized in PCR apparatus, and then set proper column temperature and detect them.2.5 Reamplification and reclaimation?. Reamplify the sample which have positive results detected by DHPLC, reclaim DNA fragment by agarose gel elec-trophoresis and gel reclaimation kit from TAKARA LTD.2. 6 DNA fragment sequencing: DNA fragments reclaimed are sequenced in TAKARA LTD.Results1. DHPLC detection : we find 11 of 17 extrons of EphB2 gene have positive results;2. DNA fragment sequencing: the sequencing results from TAKARA LTD.show that: there is 1 samesense mutations in extron6 in 8 samples, 1 mutation in intron 8 and 10 in 1 sample, respectively.ConclusionFrom our experiment results we can draw a conclusion that there is probably other mechanism between EphB2 gene and gastric cancer except gene mutation.