摘要
目的：建立非同位素银染ｍＲＮＡ差异显示方法，分离吗啡相关基因。方法：以吗啡处理的ＳＨＳＹ５Ｙ细胞中提取的总ＲＮＡ为模板，用５’ｄＴ１２ＭＧ锚定引物和两条随机引物组合进行ＤＤＲＴＰＣＲ扩增。经测序凝胶电泳分离后，用银染方法显示差异ＤＮＡ条带，在直视下从凝胶中回收差异条带并进行再扩增，扩增产物克隆入ｐＧＥＭＴ载体。结果：建立了非同位素的银染ｍＲＮＡ差异显示方法，分离和克隆了一些吗啡诱导的差异表达基因。
Objective: To develop mRNA differential display polymerase chain reaction (DD PCR) method with silver staining, isolate and clone the morphine induced genes in SH SY5Y(SY) cell line. Methods: Total RNA was extracted from the SY cells treated with morphine. The cDNA copies of differential expressed mRNA species were amplified by DD PCR using an anchored primer 5'dT 12 MG, combined with two of arbitrary primers, respectively. The DNA bands on gel were displayed by silver stain method. After running on urea denaturing sequencing gel, the differential displayed DNA bands were visually recovered from the sequencing gel and reamplified by PCR. The reamplified products were cloned into pGEM T easy vector. Results: Several morphine induced genes were identified by the silver staining mRNA differential display established. Conclusion: The developed non radioactive differential display technique with silver staining was a simple, smart, and efficient method for isolating differentially expressed gene.
出处
《北京医科大学学报》
CSCD
1998年第5期385-388,共4页
Journal of Peking University(Health Sciences)
基金
国家自然科学基金
关键词
差异显示
吗啡诱导基因
银染色法
基因扩增
MRNA
Differential display ☆ Morphine Silver staining Gene amplification RNA, messenger
