而替代了手工测序的同位素标记采用聚丙烯酰胺区分长度仅差1个碱课件.ppt

1、主要内容主要内容 ContentsContents：一、课程简介一、课程简介 核酸的分离与纯化核酸的分离与纯化 Isolation and Purification of Nucleic Acid二、电泳技术二、电泳技术 Agarose Gel Elrctrophoresis and SDS-PAGE 三、聚合酶链式反应技术三、聚合酶链式反应技术 Polymerase Chain Reaction四、四、DNADNA序列测定序列测定 DNA Sequencing五、分子杂交技术五、分子杂交技术 Molecular Hybridization Southern,Northern and West

2、ern Blot六、基因文库和六、基因文库和cDNAcDNA文库的构建文库的构建 Construction of cDNA Library and DNA Library 七、外源基因的克隆与表达七、外源基因的克隆与表达 Heterogenic Gene Cloning and Expression八、蛋白质的分离、纯化技术八、蛋白质的分离、纯化技术 Isolation and Purification of Protein Walter Gilbert Frederick Sangern1975 DNA sequencing developed: Walter Gilbert and All

3、an 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nDNADNA序列测定技术，是在高分辨率变性聚丙烯序列测定技术，是在高分辨率变性聚丙烯酰胺凝胶电泳

4、技术的基础上建立起来的。变性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聚丙烯酰胺凝胶电泳能够分离长度达到300300500500个碱基，而差别仅个碱基，而差别仅1 1个碱基的单链寡聚核苷个碱基的单链寡聚核苷酸。酸。n技术由三部分组成技术由三部分组成 1 1 产生不同长度的产生不同长度的DNADNA片段片段, ,差别仅差别仅1 1个碱基。个碱基。 2 2 在变性的聚丙烯酰胺凝胶上电泳。在变性的聚丙烯酰胺凝胶上电泳。 3 3 测序胶的放射性自显影技术。测序胶的放射性自显影技术。n末端终止法末端终止法 Sanger-Coulson (dideoxy or enzyma

5、tic) sequencingn化学裂解法化学裂解法 Maxam-Gilbert (chemical) sequencing n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。3-5 phosphodiester bondPPi (pyrophosphate)or diphosphate DNA 合成的底物合成的底物 deoxy

6、nucleoside 5-triphosphatesdNTP (dATP, dGTP, dCTP & TTP)dATPHHHHO-O-P-O-CH2OO-BASE5312HHHHO-O-P-O-CH2OO-BASE5312-O-P-O-CH2HHOHHHOOO-BASE5312533-5 Phosphodiester bridge or 3-5 phosphodiester bondDNA polymerase I fragment (Klenow), Taq DNA polymerase, Sequenase(T7 phage DNA polymerase modified)Substrat

7、e dNTP, -32p-dNTP,or -35s-dATP ddNTPdCTPddCTPHHDideoxy Sequencing of DNA proceeding for DNA synthesis: HddATPReaction Requirements forDideoxy Sequencing of DNAF1. DNA template: Single-stranded DNA molecule (template) to be sequencedF2. Primer: Oligonucleotide primer complementary to upstream region

8、of templateF3. DNA ploymerase:F4. Substrates: All four dNTPs (dATP, dGTP, dCTP, dTTP) One of dNTP is labeled with radioactive isotope. F5. One of the four ddNTPs (ddATP, ddGTP, ddCTP, or ddTTP) are needed in each reaction volume.Reaction Requirements forDideoxy Sequencing of DNADideoxy Sequencing of

9、 DNAFThe four separate sequencing ractions are loaded onto a polyacrylaimde gel and resolved by electrophoresisFThe primer or one of the four dNTPs are radioactively labeledFAn x-ray film is exposed to the gel producing an autoradiogramFThe autoradiogram is “read” from the bottom up.（一）测序反应： 1. 对于每组

10、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 5l ；pUC/M13正向引物(4.5pmol) 3.6l；无菌ddH2O至终体积16l。 3. 在引物/模板混合

11、物(以上第2步)中加入1.0l测序级Taq DNA聚合酶(5u/l)。用吸液器吸动几次混匀。4. 从第3步的酶/引物/模板混合物中吸取4l加入每一个d/ddNTP混合物的管内。5. 在微量离心机中离心一下，使所有的溶液位于eppendorf管底部。6. 把反应管放入预热至95的热循环仪，以注意中循环模式为基准，开始循环程序。每个引物/模板组合都必须选择最佳退火温度。 7. 热循环程序完成后，在每个小管内加入3l DNA测序终止溶液，在微量离心机中略一旋转，终止反应。注意 1、测序所用模板DNA的量一般按下面要求加入：模板种类/长度模板量 200bp (PCR产物) 16ng 3000-5000

12、bp（超螺旋质粒DNA) 4mg 48000bp(,粘粒DNA) 1mg 由于超螺旋质粒产生的信号比松驰的线性双链DNA弱，因此使用超螺旋质粒作为模板时其用量要比其它模板大一些。 2、计算与4.5pmol相当的引物纳克数可用以下一般公式：4.5pmol=1.5ngn, 其中n为引物碱基数 计算与1pmol相当的引物微克数可用以下一般公式： dsDNA:1pmol=(6.610-4 mg)n,n为模板碱基对数 ssDNA:1pmol=(3.310-4 mg)n, n为模板碱基数 3、为阻止Taq DNA聚合酶延伸非特异性退火引物热循环仪必须预热至95。温度变换应越快越好。下面的循环时间不包括变温

13、时间。如果你无法确定使用何种模式，建议从模式1开始。模式1：适用于引物24碱基或GC含量 C) did not work in our hands; it seems that prolonged incubation under alkaline conditions degrades the Cy-5 label; incubation of labeled DNA with NaOH for shorter periods of time is apparently not sufficient to modify DNA. MAXAM-GILBERT SEQUENCINGnThis c

14、hemical cleavage method uses double-stranded DNA samples and so does not require cloning of DNA into an M13 phage vector to produce single-stranded DNA as is the case with the Sanger-Coulson method. It involves modification of the bases in DNA followed by chemical base-specific cleavage.nStages:nDou

15、ble-stranded DNA to be sequenced is labelled by attaching a radioactive phosphorus (32P) group to the 5 end. Polynucleotide kinase enzyme and 32P-dATP is used here.nUsing dimethyl sulphoxide and heating to 90C, the two strands of the DNA are separated and purified (e.g. using gel electrophoresis and

16、 the principle that one of the strands is likely to be heavier than the other due to the fact that it contains more purine nucleotides (A and G) than pyrimidines (C and T) which are lighter).nSingle-stranded sample is split into separate samples and each is treated with one of the cleavage reagents.

17、 This part of the process involves alteration of bases (e.g. dimethylsulphate methylates guanine) followed by removal of altered bases. Lastly, piperidine is used for cleavage of the strand at the points where bases are missing.Base specificityChemical used for altered base removalChemical used forb

18、ase alterationChemical used for strand cleavageGA+GC+TAcidAcidPiperidineDimethyl suphatePiperidinePiperidineCHydrazine +alkaliPiperidinePiperidinePiperidinePiperidineHydrazineACAlkaliPiperidinePiperidinenIf reactions have been arranged to give only one, or a few, cleavages per DNA molecule, a set of

19、 end-labelled DNA fragments of different lengths is produced. nThe samples are run together on a sequencing gel which separates the fragments by electrophoresis depending on their size. DNA bands in the gel are visualized by autoradiography (32P-labelled 5 end fogs photographic film).nThe DNA sequen

20、ce is read directly from the gel. Try it yourself!nQuestions: nA. At which end of the above gel are the shortest DNA fragments? nB. What is the sequence of the sample DNA? 5-GATCGGACCT-3G G+A T+C C G G+A T+C C *GATCGGACCT*GATCGGACC*GATCGGAC*GATCGGA*GATCGG *GATCG*GATC *GAT *GA *GG 反应：硫酸二甲酯（DMS）使鸟嘌呤N7甲基化。A+G 反应：甲酸使嘌呤环上的氮质子化导致糖苷键被削弱，进而嘌呤环被吡啶取代。C+T 反应：肼能够裂解嘧啶环，进而导致其脱落。C 反应：在一定浓度的NaCl条件下，肼只对胞嘧啶起作用。在以上修饰反应完成后吡啶在加热条件下导致被修饰的碱基处磷酸二酯键断裂。