摘要
目的:分离肺腺癌及同源正常组织中的未知特异性基因片段,并建立相应的cDNA文库,为研究肿瘤基因疫苗打下良好的基础。方法:从肺腺癌及同源正常组织中分别提取总RNA,磁珠分离mRNA并逆转录为dsDNA,经RasI消化酶切将Adaptor-1和Adaptor-2R适配子分别与肺腺癌消化后的cDNA连接(作为tester),再与正常同源组织(作为driver)进行正向杂交和逆向杂交。具有上述2种不同适配子的杂交后cDNA分子,通过初次PCR和巢式PCR富集未知的cDNA片段,此PCR产物与TA克隆载体pGEM连接,转化Top10大肠杆菌,获得白色菌落并经菌液PCR扩增出未知基因片段。结果:经正向抑制性消减杂交和逆向抑制性消减杂交分别获取了肺腺癌组织及同源正常组织的特异性cDNA文库。结论:抑制性消减杂交是一种快速、方便、有效的建立cDNA文库的方法。
Objective:To isolate unknown specific gene fragment of lung adenocarcinoma tissue and homologous normal tissue and construct their corresponding cDNA library and do the groundwork for tumor gene vaccine.Methods:Total RNA were isolated from lung adenocarcinoma tissue and homologous normal tissue,from which we isolate mRNA by magnesphere technology and transcribe mRNA into ds cDNA.After ds cDNA were digested by RsaI,lung adenocarcinoma cDNA were ligated with adaptor -1and adaptor -2respectively (tester),Forward and reverse hybridize with homologous normal tissue cDNA(driver).cDNA ligated with two different adaptor after hybridization were amplified through primary PCR and nested PCR.PCR product were ligated with TA clone vector pGEM,transformated top10Escherichia coli.After white colony were obtained,unknown gene fragment were amplified by bacteria liquid PCR.Results:Specific cDNA library of lung adenocarcinoma tissue and homologous normal tissue were obtained by forward and reverse suppression subtractive hybridization.Conclusion:Suppression subtractive hybridization is a rapid?convenient ?effective method for the construction of cDNA library.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2002年第12期859-862,共4页
Chinese Journal of Clinical Oncology
关键词
抑制性消减杂交
肺腺癌
CDNA文库
Suppression subtractive hybridization Lung adenocarcinoma Homologous normal tissue cDNA library
