chap基因操作常规技术 课件.pptVIP

第四章 基因工程的常规技术;4.1 核酸的分离与检测;细胞破碎 去蛋白 DNA沉淀;CTAB法;如何保持CS-DNA的完整性?;4.1.2 质粒DNA的分离纯化;;Sol I; II; III;3. 沸水浴法;4.1.3 RNA的提取;防止RNA降解的措施;4.1.4 核酸质量检测;分子筛; 电荷效应 UV→EB→荧光;凝胶成像系统;Rf、分辨力的影响因素;缓冲液及其pH;RNA的检测;PAGE;＞40kb: Rf与分子大小无关 交替改变的电场,线团→束状;;;;影响分辨率的因素;4.2 分子杂交技术;4.2.1 探针与探针标记;5’ … G-C-T-C-A-G-C-T-G-G-A-G-T… 3’;随机引物标记;5?末端标记;2. 标记物及其检测;地高辛系统标记;生物素标记;荧光素标记;4.2.2 Southern杂交;4.2.3 Northern杂交;4.2.4 Western杂交;Semi-dry transfer system;斑点杂交;Allele-specific oligonucleotide (ASO) dot-blot hybridization can identify individuals with the sickle cell mutation. The schematic dot-blot at top shows the result of probing with an ASO specific for the normal ?-globin allele (?A-ASO). The results are positive (filled circle) for normal individuals and for heterozygotes but negative for sickle cell homozygotes (dashed, unfilled circle). The dot-blot at the bottom shows the result of probing with an ASO specific for the sickle cell ?-globin allele (?S-ASO), and in this case the results are positive for the sickle cell homozygotes and heterozygotes but negative for normal individuals. The ?A- and ?S-ASO were designed to be 19 nucleotides long in this case chosen from codons 3 to 9 of respectively the sense ?A and ?S globin gene sequences surrounding the sickle cell mutation site. The latter is a single nucleotide substitution (A→T) at codon 6 in the ?-globin gene, resulting in a GAG (Glu)→GTG (Val) substitution (see middle sequences).;制片 探针制备 杂交 镜检、拍照;基因检测新技术使膀胱癌确诊提前;4.2.7 菌落(斑)原位杂交;4.3 PCR扩增技术;4.3.1 基本原理;4.3.2 反应体系;引物的设计;耐热的DNA pol;缓冲液;4.3.2 基本过程;扩增精确性的影响因素;4.3.3 PCR技术的改进;1. 巢式PCR;2. 反向PCR;反向PCR的特点;3. Anchored PCR;特点;4. RT-PCR;5. 原位PCR;通过对PCR扩增反应中每一个循环产物荧光信号的实时监测,实现对起始模板定量分析的方法;FRET技术; 荧光标记探针 扩增、杂交、光谱、实时检测 荧光信号积累,起始模板量;起点定量与终点定量;荧光扩增曲线的三个阶段;几个参数的???定;Log浓度与循环数的关系;DNA产物的荧光标记;① SYBR Green法;;SYBR Green融解曲线分析;PCR反应体系的建立及优化;SYBR Green法优缺点;5?-R; 3?荧光淬灭基团(Q) 探针完整,R发射的荧光能量被Q吸收,无荧光; R与Q分开,发荧光 Taq酶: 5?→3?外切酶活性;;荧光共振能量转移;?扩增1条DNA→释放1个荧光分子→荧光信号的累积与PCR产物同步;TaqMan法PCR反应的建立;TaqMan法优缺点;③ 分子信标;发夹型荧光探针;荧光定量PCR的特