第三部分基因组和测序原理详解演示文稿.pptVIP

现在是30页\一共有58页\编辑于星期五 DNA template is denatured to single strands. DNA primer (with 3’ end near sequence of interest) is annealed to the template DNA and extended with DNA polymerase. Four reactions are set up, each containing: DNA template Primer annealed to template DNA DNA polymerase dNTPS (dATP, dTTP, dCTP, and dGTP) Next, a different radio-labeled dideoxynucleotide (ddATP, ddTTP, ddCTP, or ddGTP) is added to each of the four reaction tubes at 1/100th the concentration of normal dNTPs. ddNTPs possess a 3’-H instead of 3’-OH, compete in the reaction with normal dNTPS, and produce no phosphodiester bond. Manual Dideoxy DNA sequencing -How it works: 现在是31页\一共有58页\编辑于星期五 Whenever the radio-labeled ddNTPs are incorporated in the chain, DNA synthesis terminates. Each of the four reaction mixtures produces a population of DNA molecules with DNA chains terminating at all possible positions. Extension products in each of the four reaction mixutes also end with a different radio-labeled ddNTP (depending on the base). Next, each reaction mixture is electrophoresed in a separate lane (4 lanes) at high voltage on a polyacrylamide gel. Electrophoresis at 70°C (2 h). Fix gel in HAc, dry gel (1 h). Autoradiography (24-48 h). Pattern of bands in each of the four lanes is visualized on X-ray film. Location of “bands” in each of the four lanes indicate the size of the fragment terminating with a respective radio-labeled ddNTP. DNA sequence is deduced from the pattern of bands in the 4 lanes. 现在是32页\一共有58页\编辑于星期五 取代放射性同位素的方法 地高辛,生物素: 用地高辛,生物素代替发射性同位素示踪测序反应产物,最后用酶显色反应显示出测序结果. 银染方法: 把生成DNA分子核苷酸碱基顺序的方法与一种超敏感的银染方法相结合。这一新的技术组合使电泳分离的序列片段不依赖于仪器就直接可见。这种无放射性的系统包括通过用酶促双氧链终止反应形成一系列DNA序列片段，凝胶电泳分离这些片段，并把凝胶浸入超敏感的银染溶液中,观察引物延伸产物的银染条带,从而确定DNA分子的序列。 现在是33页\一共有58页\编辑于星期五 荧光标记物 ddGTP ddATP ddTTP ddCTP 现在是34页\一共有58页\编辑于星期五 引物标记法测序(Dye Primer) 一个样本，4个反应。4个反应中引物序列相同，但分别标记有不同颜色的荧光。 + AmpliTaq FS enzyme dCTP, dATP, dGTP, dTTP ddATP (terminator) + AmpliTaq FS enzyme dCTP, dATP, dGTP