| Microsatellite is the simple serial repetitive DNA sequence widely distributed in the genome of prokaryocytes and eucaryotic cells with high degree of polymorphism and very low variation rate, showing the Mendel's codominant inheritance. The genesis and development of a variety of human tumors has relation to the heredity instability of specific micro satellite (MSI) DNA in genome and loss of heterozygosity (LOH) in the corresponding allelic gene; and LOH of micro satellite in some specific chromosome are usually associated with the inactivation of cancer inhibitory gene at the corresponding site which plays an important role in the genesis and development of tumors. It has been discovered that the phenomenon of MSI is widely present in many sporadic tumors such as leukemia, cancer of the bladder, melanoma, glioma, meningioma, and neuroblastoma. It has been demonstrated that the higher eucaryotic cell has mismatch repair gene system (MMR), which participates in the process of repair after replication to maintain the stability and fidelity of DNA replication by eliminating the error of DNA synthesis. At present the known MMR system includes mainly 6 kinds of gene, namelyhMLH1, hMSH2, hMSH3, hPMS1, hPMS2 and hMSH6. Should the MMR and gene function be inactivated (e.g. mutation of gene, methylation of promoter region), the mistake in the process of DNA replication would not be corrected, which may lead to the instability of micro satellite DNA repetitive sequence, manifesting as MSI and LOH. Therefore, the study on MSI in glioma is helpful for understanding the mechanism underlying the genesis and development of glioma and for revealing pathogenesis of glioma on the molecular basis.Experimental Materials and Instruments1. Experimental materials: Astrocytoma specimen were obtained from the specimen removed during surgery at the Dept. of Neurosurgery of Second Affiliated Hospital of Medical College, Zhejiang University. The peripheral venous blood were drawn at the same time for normal control. Reagents: protease K, Taq DNA polymerase were purchased from DAKO Company, Denmark; Chloroform, isoamyl alcohol, ethanolamine, ethyl alcohol and sodium acetate were bought from Hangzhou Changzheng Chemical Reagent Factory.2. Experimental instruments: Thermostatic charmer was bought from Ningbo Jiangnan Instrument Factory; PCR electrophoresis apparatus and ultra-speed centrifuge, from Beijing Liuyi Instrument Factory; Ultraviolet spectrophotometer, from Shanghai Analytical Instrument Factory.Experimental Methods1. Extraction of DNA(1) DNA-extraction from the histo-specimen; The specimen is digested with protease K after being crushed, then shaking for 12-18 hr at 50 . Using equal volume of phenol / chloroform / isoamyl alcohol for the extraction of DNA; precipitating with ethanolamine and ethyl alcohol; and dissolving with TE, then keeping the producer at 4癈.(2) DNA-extraction from blood specimen; The specimen is digested with protease K, then set at 50癈 water bath for 1 hr. Using phenol / chloroform / isoamyl alcohol to extract the DNA and then precipitating with absolute ethyl alcohol and sodium acetate, dissolving in TE and keeping the producer at 4 .2. The amplification of microsatellite PCRSelecting the microsatellite site at D9S171, D9S165, D9S54, APOC2, D19S112, HRC respectively for the assay. The information of micro satellite DNA primer sequence was obtained from Genome Data Base (http://gdbwww.gdb.org/gdb), the primer was synthesized by the Institute of Cytobiology of Chinese Academy of Sciences, Shanghai. The PCR reaction system are 50mmol/L KC1, 10mmol/L Tris-Cl 1.5mol/L MgCl2 200 u mol/L dNTPs template DNA 200ng, Upstream and downstream primer each of 50 pmol, Taq DNA polymerase lu added with water to 50 uL. The PCR reaction parameter: degeneration for 5min at 94 , then entering circulation (94 50 sec, 55 60 sec,72 60 sec) for a total of 35 circulations, then for extension at 72癈 for 10min and keeping the producer at 4 .3.
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