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Lecture 28

Lecture 28. 附录 D 实验技术 D.1 DNA 操作技术 D.2 操作蛋白质 D.3 基因及其 蛋白质的功能. Appendix D Experimental Techniques D.1 DNA manipulation techniques D.2 Manipulating Proteins D.3 The Function of Genes and their Proteins. Appendix D Experimental Techniques / 实验技术.

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Lecture 28

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  1. Lecture 28 附录D 实验技术 D.1 DNA操作技术 D.2 操作蛋白质 D.3 基因及其 蛋白质的功能 Appendix D Experimental Techniques D.1 DNA manipulation techniques D.2 Manipulating Proteins D.3 The Function of Genes and their Proteins

  2. Appendix DExperimental Techniques / 实验技术 www.entu.cas.cz/jindra/ www.cellmed.com/html/facilities.htm

  3. D.1 DNA manipulation techniques DNA操作技术 D.1.1 Gel Electrophoresis and Southern Blotting D.1.2 Polymerase Chain Reaction (PCR) D.1.3 Recombining DNA D.1.4 DNA sequencing D.1.5 Molecular Cloning

  4. D.1.1 Gel Electrophoresis and Southern Blotting凝胶电泳与Southern印迹法

  5. Now showingSeparating Fragments of DNA by Gel Electrophoresis File: biophoto7 \ Life \ Separating fragments of DNA by gel electrophoresis (10’)

  6. Southern Blotting / Southern印迹法

  7. 补充介绍：DNA鉴定 1. DNA fingerprints DNA指纹 2. DNA fingerprinting DNA指纹分析 3. DNA typing DNA分型 4. An example of DNA identification DNA鉴定实例

  8. 1. DNA fingerprints / DNA指纹 Every person’s fingerprints are different. Fingerprints / 普通指纹 • Method to distinguish: • Take photographes; • Compare. Every person’s DNAs are different. DNA fingerprints / DNA指纹 Method to distinguish: DNA fingerprinting

  9. Difference of DNAs / DNA的不同之处 The difference of human DNAs comes from different locations of minisatellite DNAs. （人DNA之间的不同来源于小卫星DNA所处的位置不同。） Gene X Gene Y Person 1 Gene X Gene Y Person 2 Minisatellite DNA: a short sequence of 12 or more base pairs repeated many times in tandem. (小卫星DNA：由12bp或更多一点的短序列以串联方式重复多次形成的DNA.)

  10. 2. DNA fingerprinting / DNA指纹分析 (1) Cut with HaeIII （酶切） C A B (2) Gel electrophoresis （凝胶电泳） A B C

  11. DNA fingerprinting (continued) DNA指纹分析（续） A B C (3) Southern blotting (Southern转印) Person 1 Person 2 A A B B C C B C ( ) (4) Hybridize to labeled minisatellite DNA; (5) Detect label with X-ray film 与标记的小卫星DNA杂交；用X光片检测

  12. DNA fingerprint / DNA指纹 DNA fingerprint: a picture showing the variable regions of DNA for identifying particular individuals. DNAs from unrelated persons（无血缘关系的个体） Twin’s DNAs （双胞胎） （DNA指纹：指显示了不同DNA区域可用于鉴定特殊个体的图片。） 1 2 3 4 5 6 7 8 9 10 11

  13. 3. DNA typing /DNA分型 DNA typing: the analysis of DNA using probes that hybridize to fewer DNA bands. （DNA分型：使用与较少DNA条带杂交的探针对DNA进行分析的技术。） Marker 嫌犯A / Suspect A 罪犯血迹/ Criminal’s blood 嫌犯B / Suspect B Marker 罪犯头发/ Criminal’s hair 被害人/ Victim Control DNA Marker No DNA

  14. 探针个数与DNA鉴定准确率 百里挑一 一个探针可以对一般人群中的100人加以区分。 (A given probe detects a set of bands in one in a hundred people in the general population. ) Number of probes 探针个数 Misjudgment Possibility 错判可能性 Identifying accuracy 鉴定准确度 1 2 3 4 5 10-2 10-4 10-6 10-8 10-10 99% 99.99% 99.9999% 99.999999% 99.99999999%

  15. 4. DNA鉴定实例 DNA鉴定：使用DNA分型技术对特殊个体身份进 行鉴定的过程。 (DNA identification: A procedure employing DNA typing technique to identify an specific individual.) Have we got him? 真假萨达姆

  16. D.1.2 Polymerase Chain Reaction (PCR) 聚合酶链式反应

  17. Polymerase Chain Reaction

  18. Polymerase Chain Reaction

  19. Now showingPolymerase chain reaction File: biophoto7 \ Introduction to Genetic Analysis \ Polymerase chain reaction (5’).

  20. Polymerase Chain Reaction (1/9) A sample of chromosomal DNA, also called geno-mic DNA, can be used as the starting material for polymerase chain reaction (PCR). With PCR, an investigator can amplify a single copy of a DNA segment into billions of identical copies.

  21. Polymerase Chain Reaction (2/9) In addition to the target DNA, a PCR reaction contains several other ingredients. These include free nucleotides, DNA primers, and the enzyme Taq polymerase. The primers are typically about 20 nucleotides long and are complementary in sequence to the ends of the target DNA. Taq polymerase is derived from hot springs bacteria and can tolerate the intense heat of a PCR reaction.

  22. Polymerase Chain Reaction (3/9) A PCR reaction lasts several hours and typically consists of 20 to 35 repeating cycles. A cycle begins by heating the reaction mixture to 95 degrees Celsius. The heat denatures the DNA, breaking the hydrogen bonds that hold the strands together.

  23. Polymerase Chain Reaction (4/9) After denaturing the DNA, the temperature is reduced to around 60 degrees so that the primers can form hydrogen bonds, or anneal, with their complementary sequences in the target DNA. Note that the primers and the target DNA follow base-pairing roles. An adenine (A) pairs with a thymine (T), and a cytosine (C) pairs with a guanine (G).

  24. Polymerase Chain Reaction (5/9) In the next phase, the temperature is raised to 72 degrees Celsius. Taq polymerase functions optimally at this temperature and begins polymeri-zation, adding nucleotides to the 3’ end of each primer attached to a DNA strand. After one complete cycle, there are two double-stranded copies of the target DNA.

  25. Polymerase Chain Reaction (6/9) The PCR reaction mixture contains many copies of the primers and an abundant supply of nucleotides to perform many addition cycles. After a second cycle, there are four copies of the target DNA.

  26. Polymerase Chain Reaction (7/9) After cycle 3 is finished, there are eight copies of the double-stranded target DNA sequence. Note that only two of the double-stranded copies consist of just the target fragment. The others also include flanking DNA regions.

  27. Polymerase Chain Reaction (8/9) As the number of cycles increases, the products consist of a greater proportion of fragments with just the target DNA. After four cycles, half of the fragments consist of just target DNA, and half of the fragments also contain flanking DNA.

  28. Polymerase Chain Reaction (9/9) With each additional cycle, the number of copies of our target sequence doubles. At the end of cycle 25 there are more than 33 million copies of this double stranded target region.

  29. Now showing again

  30. PCR is used to copy specific region of DNAPCR用来拷贝DNA的特殊区域

  31. D.1.3 Recombining DNA / 重组DNA

  32. Ligation of DNA fragmentsDNA片段的连接

  33. D.1.4 DNA sequencing / DNA序列测定

  34. DNA sequencing / DNA序列测定

  35. Now showingDideoxy sequencing of DNA File: biophoto7 \ Lehninger Principles of Biochemistry \ Dideoxy sequencing of DNA (5’).

  36. DNA sequencing (1/6) A short, radiolabeledprimer is annealed to the single-stranded DNA to be sequenced. The DNA serves as a template for in vitro DNA synthesis. The DNA-primer mixture is split into four separate tubes. DNA polymerase and a solution of dNTPs are added to each tube.

  37. DNA sequencing (2/6) One of the four 2’,3’ dideoxy-NTPs (ddNTPs) is mixed into each tube, at a concentration 100 times lower than the dNTP concentration.

  38. DNA sequencing (3/6) DNA polymerase uses the dNTPs to elongate the primer DNA. The occasional incorporation of a ddNTP terminates the polymerization reaction, since the absence of a 3’ hydroxyl blocks addition of the next nucleotide.

  39. DNA sequencing (4/6) The ddNTPs are incorporated randomly, resulting in terminated fragments of different lengths.

  40. DNA sequencing (5/6) The terminated fragments from each of the four reactions are denatured and separated by size using gel electrophoresis.

  41. DNA sequencing (6/6) The gel is autoradiographed in order to visually detect the labeled DNA fragments. The resulting ladder is read from bottom to top. This represents the complementary sequence of the original DNA template.

  42. Now showing again

  43. D.1.5 Molecular Cloning / 分子克隆

  44. Molecular Cloning / 分子克隆

  45. Now showingPlasmid Cloning File: biophoto7 \ Lehninger Principles of Biochemistry \ Plasmid cloning (8’)

  46. Lecture 29 附录D 实验技术 D.1 DNA操作技术 D.2 操作蛋白质 D.3 基因及其 蛋白质的功能 Appendix D Experimental Techniques D.1 DNA manipulation techniques D.2 Manipulating Proteins D.3 The Function of Genes and their Proteins

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