变性聚丙烯酰胺凝胶电泳能够分离长度达到300～500个碱基.PPTVIP

DNA序列测定DNA sequencing Walter Gilbert Frederick Sanger 1975 DNA sequencing developed: Walter Gilbert and Allan Maxam of Harvard University and Fred Sanger of Cambridge University simultaneously come up with two techniques for determining the exact sequence of bases that make up a gene. Gilbert and Sanger share the 1980 Nobel Prize (also with Paul Berg). 一、概述 DNA序列测定技术，是在高分辨率变性聚丙烯酰胺凝胶电泳技术的基础上建立起来的。变性聚丙烯酰胺凝胶电泳能够分离长度达到300～500个碱基，而差别仅1个碱基的单链寡聚核苷酸。 技术由三部分组成 1 产生不同长度的DNA片段,差别仅1个碱基。 2 在变性的聚丙烯酰胺凝胶上电泳。 3 测序胶的放射性自显影技术。 末端终止法 Sanger-Coulson (dideoxy or enzymatic) sequencing 化学裂解法 Maxam-Gilbert (chemical) sequencing 末端终止法原理 利用E.coli polymerase I以单链DNA为模板，以dNTP和[?-32p]-dATP为底物合成互补的DNA链。当反应遇到双脱氧的核糖核苷酸底物（ddNTP）时，掺入到新生的DNA链中，但是该双脱氧的核糖核苷酸的掺入阻止了DNA链进一步的延伸反应，形成了长短不同的DNA片段。通过电泳和放射自显影读出DNA序列。 Phosphodiester bonds in DNA DNA sequencing Dideoxy Sequencing of DNA Reaction Requirements forDideoxy Sequencing of DNA 1. DNA template: Single-stranded DNA molecule (template) to be sequenced 2. Primer: Oligonucleotide primer complementary to upstream region of template 3. DNA ploymerase: 4. Substrates: All four dNTPs (dATP, dGTP, dCTP, dTTP) One of dNTP is labeled with radioactive isotope. 5. One of the four ddNTPs (ddATP, ddGTP, ddCTP, or ddTTP) are needed in each reaction volume. Reaction Requirements forDideoxy Sequencing of DNA Dideoxy Sequencing of DNA The four separate sequencing ractions are loaded onto a polyacrylaimde gel and resolved by electrophoresis The primer or one of the four dNTPs are radioactively labeled An x-ray film is exposed to the gel producing an autoradiogram The autoradiogram is “read” from the bottom up. （一）测序反应： 　　　 　1. 对于每组测序反应，标记四个0.5ml eppendorf管(G、A、T、C)。每管加入2ml适当的d/ddNTP混合物(d/ddNTP Mix)。各加入1滴(约20ml)矿物油，盖上盖子保存于冰上或4℃备用。 　　 　2. 对于每组四个测序反应,在一个eppendorf管中混合以下试剂：（1） 样品反应：质粒模板DNA 2.1pmol；5×测序缓冲液 5ml；引物 4.5pmol；无菌ddH2O 至终体积16ml 。 （2）对照反应：pGEM-3Zf(+)对照DNA(4mg)　 4.0 ?l；5×测序缓冲液 5?l ；pUC/M13正向引物(4.5pmol) 3.6?l；无菌d