ch2-2-工具酶-OTHER ENSYMESnew.pptVIP

（2）??匹配接头的种类 i.?? 预制匹配接头(连接平端和粘性末端) 人工接头 + 匹配接头 预制匹配接头 Linker ii. 转换匹配接头(连接5’-突起和3’-突起末端) 二匹配接头退火 转换匹配接头 双酶切 （3） 匹配接头连接法 转换匹配接头 EcoR I Mse I 2. 用Klenow补齐3’凹端或部分补齐3’凹端。 TCGA AGCT 载体 Xho I 外源DNA Sau3A I GATC CTAG AGCT TCGA TC CT Klenow dCTP dTTP CTAG GA AG GATC Klenow dATP dGTP TCGA TC CT AGCT GATC dATP dGTP GATC AG GA CTAG T4 DNA LIGASE 3. 用核酸酶、Klenow酶、T4 DNA pol去除3’突出末端产生平端。 4. 用末端转移酶，在载体和外源DNA的酶切点的3’端加上互补的同聚尾。 4. 对于5’突出末端，现用Klenow补齐3’凹端，再用末端转移酶，在载体和外源DNA的3’端加上互补的同聚尾。 4. 对于平头末端，用末端转移酶在载体和外源DNA的3’端加上互补的同聚尾。 TTTTT XhaI TTTTT TTTTT AAAAA TTTTT AAAAA XhaI XhaI 反转录酶 dNTP RNA酶，DNA聚合酶 T4DNA聚合酶 寡聚dT引物 寡聚dT＋转接头引物 RNA酶，DNA聚合酶 T4DNA聚合酶 EcoR I转接头 T4DNA连接酶 XhaI酶 T4DNA连接酶 EcoR I转接头 EcoR I EcoR I EcoR I 载体 T4DNA连接酶 5. 利用PCR技术 THIS IS END OF CHAPTER 2 * * * * * in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (ApopTaq peroxidase kit, Intergen Company, Purchase, NY) of neurons undergoing apoptotic cell death (brown-colored nuclear staining). A. Cerebellar cortex, showing occasional positively stained Purkinje cells. B. Hippocampus. C. Positively stained neurons within a microglial nodule. D. Positively stained neurons in cerebral cortex. Magnification 200x. * Detection of self-spliced and unspliced luciferase RNAs in 293 cells. (A) RNase protection assay to distinguish spliced RNA (228 nucleotide protection product) from unspliced precursor (146 nucleotide + 82 nucleotide protection products). Thick black bars, luciferase RNA exons; thin black loop, intron; gray bars, RNase protection probe. A separate probe was synthesized for each intron position. (B) Titration of increasing amounts (0.0078–2.0 g) of RNA from Luc(958)-transfected cells. Following RNase protection analysis, the products were analyzed by 4% polyacrylamide–7 M urea gel electrophoresis. Lane +, unhybridized probes digested with RNases A and T1; lane -, 278 nucleotide luciferase probe plus 400 nucleotide -gala