DNA length polymorphsms苏州大学法医系.ppt

DNA length polymorphisms 苏州大学法医系 Xiashuixiu@suda.edu.cn DNA Polymorphisms Sequence polymorphism ….AGACTAGACATT….. ….AGATTAGGCATT….. Length polymorphism ….AATGAATGAATG…. ….AATGAATG….. RFLP基本技术 DNA的提取 RE消化 琼脂糖电泳分离 印迹转移 探针选择 探针的标记 分子杂交 谱带显示 Step 1 DNA的提取 DNA的纯化 有机溶剂萃取、乙醇沉淀 柱层析过滤法 凝胶柱层析技术 DNA 的定量 琼脂糖凝胶电泳结合溴乙锭荧光测定法 紫外分光光度法 DNA探针杂交法 Step 2 RE 消化 Restriction Enzymes Restriction endonucleases cut DNA molecules at defined positions Digestion of DNA with different restriction endonucleases Step 3 电 泳 电 泳 琼脂糖凝胶电泳 100 bp ~ 60 kb 的大片断DNA 聚丙烯酰胺凝胶电泳 5 ～500 bp 的小片断DNA Step 4 印迹转移 Step 5 探针的选择 DNA 探针 DNA 探针是一条较短的已知序列的DNA单 链。RFLP探针是小卫星DNA的核心序列串 联重复的多聚体片断 DNA 纹印 Step 6 探针的标记 探针的标记 同位素标记法 非同位素标记法 半抗原类标记法 生物素类标记法 酶标法 Step 7 分子杂交 分子杂交 概念：特异性探针与待测DNA 在复性条件下形成稳定异源DNA双链的过程 步骤 预杂交： 杂交 离子强度：以Na离子中和DNA的负电荷 探针浓度： 探针长度 杂交温度 洗膜 多基因座探针采用5×SSC液 单基因座探针采用0.1×SSC液 1×SSC液：0.15mol/L氯化钠和0.015mol/L柠檬酸钠 Step 8 谱带显示 X光片感光 定影 显影 Amp-FLP Amp-FLP Amplified Fragment Length Polymorphisms Short Tandem Repeats(STRs) STR Allele Frequencies PCR 技术 Mutations Mutations can occur at STR loci Most STR mutations involve the gain or loss of a single repeat unit. Can be difficult to determine from which parent contributed the mutant allele. Low mutation rates are important for paternity testing. Chemical Inhibition of Enzymes Materials such as textile dyes or hemoglobin can inhibit enzymatic function. Results can look like degraded DNA. Solutions to PCR Inhibition Remove the inhibitor by re-purifying the DNA sample. Dilute the effects of the inhibitor by: Adding less sample volume; Adding more DNA polymerase; Adding bovine serum albumin (BSA); In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created PCR Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Thermal cycle Three Steps Denaturation Annealing Extention Step 1: denaturation Combine Target Sequence, DNA primers template, dNTPs, TAQ Polymerase Target Seque