Chapter 20 Techniques of Molecular Biology

1. Chapter 20 Techniques of Molecular Biology

2. molecular biology means the understanding organism from biology. It is just a tool ,but ,it is the most import tool for current research The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves. foreword

3. Ⅰ.gain ＆identification Ⅱ.Sequencing ＆structural analysis Ⅲ.functional analysis Ⅳ.informational integration

4. 注：余不苟同Watson诸人以操作之对象来划分章节也。窃以为，参考分子操作的具体步骤可以更好地理解分子生物学技术本身哉。余虽浅陋，然不愿轻弃己意——遂有此PPT。注：余不苟同Watson诸人以操作之对象来划分章节也。窃以为，参考分子操作的具体步骤可以更好地理解分子生物学技术本身哉。余虽浅陋，然不愿轻弃己意——遂有此PPT。

5. Ⅰ.gain ＆identification

6. gain a). Isolation of special genes b). Gain and purification of proteins c). RNA separation

7. a). Isolation of special genes Techniques: DNA cloning ＆ PCR “A golden era of molecular biology was launched once it became possible to isolate specific DNA segments representing individual genes .” ----《Molecular Biology of Genes》

8. DNA Cloning • “The ability to construct recombinant DNA molecules and maintain them in cells is called DNA Cloning. ” ----《Molecular Biology of Genes》 • Topically involves a vector : the most common one is a plasmid.

9. Characteristics of vector DNAs: • 1.an origin of replication • 2.a selectable marker • 3.sigle sites for one or more restriction enzymes.

10. Construction of a genomic DNA library: • Genomic DNA and vector DNA, digested with the same restriction enzyme, are incubated together with ligase • The resulting pool or library of hybrid vectors is then introduced into E. coli, and the cells are plated onto a filter placed over agar medium. • The filter is removed from the plate and prepared for hybridization.

12. Construction of a cDNA library • Isolate mRNA • use reverse transcriptase to synthesize complementary DNA strand from mRNA, then use DNA Pol I to synthesize double stranded DNA. Clone these cDNAs into appropriate vector (usually plasmid or phage) • Use Oligo dT primer to hybridize to polyA tail of mRNA. Primer used by reverse transcriptase for extension. • Reverse transcriptase is a DNA polymerase which uses RNA as a template to synthesize complementary DNA. Cloned from RNA viruses.

13. Construction of a cDNA library

14. Difference between of a genomic DNA library and a cDNA library

15. PCR • PCR: polymerase chain reaction .A powerful method for amplifying particular srgments of DNA ,which is carried out entirely biochemically in vitro. • Elements: 1.Template ; 2.The four nucleotides ---dNTPs ; 3.The enzyme DNA polymerase 4. A primer.

16. PCR

17. Typical steps of PCR • Denaturation at 94℃ : the double strand melts open to single stranded DNA, all enzymatic reactions stop . • Annealing at 54℃ :The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. • Extension at 72℃ :This is the ideal working temperature for the polymerase. The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

18. Something else : Site-directed mutagenesis Site-directed mutagenesis :A custom-designed oligonucleotide harboring a mismatch to a segment of cloned DNA can be used to create a directed mutation in that cloned DNA . A synthetic DNA fragment is used as a tool for changing one particular code word in the DNA molecule. This reprogrammed DNA molecule can direct the synthesis of a protein with an exchanged amino acid.

19. b). Gain and purification of proteins • Techniques: Electrophoresis ＆ • Chromatography • Basis : • To purify proteins we make use of their inherent similarities and differences. • Protein similarity is used to purify them away from the other non-protein contaminants. • Differences are used to purify one protein from another. Proteins vary from each other in size, shape, charge, hydrophobicity, solubility, and biological activity.

20. chromatography • In this approach, protein fractions are passed though glass columns filled with appropriated modified small acrylamide or agarose beads. • There are various ways columns can be used to separate proteins according to their characteristics. • Ion exchange chromatography, gel filtration chromatography, Affinity Chromatography ,and so on

21. fig 1 principle of Ion exchange chromatography

22. Fig 2 Principle of gel filtration chromatography

23. Fig 3 Principle ofAffinity Chromatography

24. antibodies visualize electrophoretically-separated proteins. • The electrophoretically separated proteins are transferred to a filter. And this filter is then incubate in a solution of an antibody to our interested protein. Finally, a chromogenic enzyme is used to visualized the filter-bound antibody

25. identification

26. DNA gel electrophoresis Linear DNA molecules migrate through the gel toward the positive pole with different rates according to different size

27. pulsed-field gel electrophoresis • Pulsed-field gel electrophoresis forlong DNAs (up to severalMb in length).

28. Restriction Endonuleases Cleaves DNA Molecules at Particular Sites Recognition sequences and cut sites of various endonucleases

29. Southern blot 1. DNA fragments, generated by digestion of a DNA molecule by a restriction enzyme, are run out on an agarose gel. 2. Once stained, a pattern of fragments is seen. 3.When transferred to a filter and probed with a DNA fragment homologous to just one sequence in the digested molecule, a single band is seen, corresponding to the position on the gel of the fragment containing that sequence.

31. Varieties of blots

32. Ⅱ.Sequencing ＆structural analysis

33. Outline of this part

34. Sequencing of DNA and genome Techniques : Ⅰ. DNA molecules (radioactively labeled at 5’ termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately. Ⅱ. chain-termination method. The second one is more current .

35. The absence of 3’-hydroxyl lead to the inefficiency of the nucleophilic attack on the next incoming substrate molecule

36. Techniques Ⅱ. chain-termination method • DNA molecules (radioactively labeled at 5’ termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately.

37. Techniques Ⅱ. chain-termination method 4 systems with dNTP+ ddGTP, dNTP+ ddATP d NTP+ ddCTP, d NTP+ ddTTP separately. And even ,we can “read” the sequencing gel to get the sequence of the DNA

38. fluorescent chain-terminating nucleotides With the method of fluorescent chain-terminating nucleotides, we can carry out high throughput sequence

39. Sequencing a genome • Question: chain-termination method is powerful ,however, it can only sequence a fragment of 800bp as most at time. • Solution: the shotgun sequencing ,

40. the shotgun sequencing

41. The shortgunstrategy permits a partial assembly of large genome sequence

42. read containing identical sequences are assumed to overlap and are joined to form larger contigs .And paired-end reading leads to form scaffold .

43. StructuralanalysisofDNA The two strands twisting around each other in the form of a double helix. base pairs : adenine-thymine guanine-cytosine

44. Base pairs

45. Schematic model Space-filling model

46. Importance It is the reveal of the structure of DNA that bring the golden era of molecular biology It reveal how DNA carries genetic messages and how DNA reply .

47. Sequencing of protein • Techniques: • 1. Edman degradation; • 2. MS/MS

48. Edman degradation The N-terminal residue is labeled and can be removed without hydrolyzing the rest of the peptide. Thus, in each round, one residue is identified, and that residue represents the next one in the sequence of the peptide.

49. Tandem mass spectrometry Material travels through the instrument in a manner that is sensitive to its mass/charge ratio. It is a method with great accuracy.

50. Structural analysis of protein • Techniques: • 1.NMR; • 2.X-ray diffraction; • 3.Crystalization.