Isolate The Genes Related To The Colorectal Carcinoma With Suppression Subtractive Hybridization

The colorectal tumor is the frequent malignant tumor in digestive tract in China, and the mortality of colorectal tumor is increasing this years. Occurrence of tumor results from activation of pro-oncogene and inactivation of tumor suppressor gene. Today, we have not discovered the specific genes which are close associative with the development of colorectal cancer, thought we have learned that there are much changes of genes in the development of colorectal cancer. Therefore , it is no effective method to diagnose , treat the pristine colorectal cancer. With the quickly development of molecular biology technique, it is very possible to discover the genes which is close associated with the colorectal cancer, and deeply study the molecular mechanism of colorectal cancer.Object: To construct subtracted cDNA libraries of colorectal carcinoma and normal intestine tissue using the technique of Suppression Subtractive Hybridization(SSH). Isolate and clone the genes which are associated with the colorectal cancer, in order to clarify the molecular mechanism of colorectal cancer. And provide the basic theory for diagnosis and gene treatment in the pristine colorectal cancer.Methods: Take the colorectal carcinoma and normal intestine tissue as studyobjects. Isolate the total RNA from two tissue and purify into mRNA, then become the cDNA with reverse transcription. Take the colorectal cancer cDNA as the Tester cDNA and normal intestine tissue as the Driver cDNA. After digested with the Rsal, the digested cDNA ligated with adaptors, then two steps of hybridization and two round of PCR were followed to normalize and enrich differently expressed genes. Ligate the product of Nest PCR with T-easy vector the transform the coli competent cell DH5ct to construct subtracted cDNA libraries of colorectal carcinoma and normal intestine tissue. Identify and clone the positive clones. Then sequence the differently expressed genes and analyzed with BLAST program in GenBank wired NCBI website. Some sense genes were identified by Virtual northern blot.Results: Total RNA and mRNA were respectively exacted and purified from the colorectal cancer and normal intestine tissue in good quality and quantity. Double strand cDNA were reversing transcripted integrally, and then cut by Rsal into even length short segments. Ligation was effective. After two stage of subtractive hybridization and Nest-PCR, the differently expressed cDNA were obtained and enlarged. Subtractive hybridization was effective. After constructing subtracted cDNA library of colorectal carcinoma and normal intestine tissue, 200 clones were randomly picked from the library and identified by PCR. The results showed that 176 differently expressed cDNA were inserted successfully. 50 positive clones were selected for DNA sequencing and analyzed with BLAST program in GenBank wired NCBI website and 20 differently express genes were obtain. Six sense genes were identified by Virtual Northern and the results showed the reliability of this study.Conclusions: The success fully constructed subtracted cDNA library of colorectal carcinoma and normal intestine tissue and 20 differently expressed genes were found. The characterization of these genes contained pro-oncogene, synthesis of DNA, cell signaling, energy metabolism, cell apoptosis. And 2 new genes related tothe colorectal cancer were found.