Molecular Biology Course Outline 2007

1. Molecular Biology Course Outline 2007 Book: Molecular Biology, 4th edition, McGraw Hill by Robert Weaver Molecular and Cell Biology I (Wednesdays 3:00 -6:00 pm) 由謝明麗,劉薏雯,蔡世峰教授合授 Week Date Topic Instructor 1 9/19 Molecular tools for studying Gene activity 謝明麗 2 9/26 Molecular tools for studying Gene activity (II) 謝明麗 3 10/3 The transcription apparatus of Prokaryotes 謝明麗 4 10/10 Operons: Major shift in Prokaryotic transcription 謝明麗

2. 5 10/17 Eukaryotic RNA polymerases and their promoters 劉薏雯 • 6 10/24 General transcription factors in Eukaryotes 劉薏雯 • 7 10/31 Transcription activators in Eukaryotes 劉薏雯 • 8 11/7 Message RNA processing: Splicing 劉薏雯 • 9 11/14 Message RNA processing: Capping and polyadenylation 劉薏雯 • 10 11/21 Mid-term 劉薏雯 • 11 11/28 The Human Genome Project and the HapMap Project 蔡世峰 • 12 12/5 Genomic technology, microarray, and proteomics 蔡世峰 • 13 12/12 Cancer genomics, microbial genomics, and pharmacogenomics 蔡世峰 • 14 12/19 Model organisms and systems biology 喻秋華 • 15 12/26 特別演講 • 16 1/2 特別演講 • 17 1/9 Overview

3. Molecular Tools for Studying Genes and Gene Activity

4. Molecular Separation

5. Gel Electrophoresis • Gel electrophoresis is used to separate different species of: • Nucleic acid • Protein

6. DNA Gel Electrophoresis • Melted agarose is poured into a form equipped with removable comb • Comb “teeth” form slots in the solidified agarose • DNA samples are placed in the slots • An electric current is run through the gel at a neutral pH

7. DNA Separation by Agarose Gel Electrophoresis • DNA is negatively charged due to phosphates in its backbone and moves to anode, the positive pole • Small DNA pieces have little frictional drag so move rapidly • Large DNAs have more frictional drag so their mobility is slower • Result distributes DNA according to size • Largest near the top • Smallest near the bottom • DNA is stained with fluorescent dye

8. DNA Size Estimation • Comparison with standards permits size estimation • Mobility of fragments are plotted v. log of molecular weight(or number of base pairs) • Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation • Same principles apply to RNA separation

9. Electrophoresis of Large DNA • Special techniques are required for DNA fragments larger than about 1 kilobases • Instead of constant current, alternate long pulses of current in forward direction with shorter pulses in either opposite or sideways direction • Technique is called pulsed-field gel electrophoresis (PFGE)

10. Protein Gel Electrophoresis • Separation of proteins is done using a gel made of polyacrylamide (polyacrylamide gel electrophoresis = PAGE) • Treat proteins to denature subunits with detergent such as SDS • SDS coats polypeptides with negative charges so all move to anode • Masks natural charges of protein subunits so all move relative to mass not charge • As with DNA smaller proteins move faster toward the anode

12. Summary • DNAs, RNAs, and proteins of various masses can be separated by gel electrophoresis • Most common gel used in nucleic acid electrophoresis is agarose • Polyacrylamide is usually used in protein electrophoresis • SDS-PAGE is used to separate polypeptides according to their masses

13. Two-Dimensional Gel Electrophoresis

14. Ion-Exchange Chromatography • Uses a resin to separate substrances according to their charges • DEAE-Sephadex chromotography uses an ion-exchange resin that contains positively charged diethylaminorthyl (DEAE) group. • These positive charges attract negatively charged substances, including proteins. • Phosphocellular is commonly used negatively charged resin.

16. Gel Filtration Chromatography • Uses columns filled with porous resins that let in smaller substances, but exclude larger ones. • The smaller substances are slowed in their journey through the column, but larger substances travel relatively rapidly through the column.

18. Tracers Detection Labeled tracers (e.g. 3H, 14C, 32P, 35S, 125I) Autoradiography using x-ray film phosphorimaging, liquid scintillation counting Non-radioactive tracers (e.g. fluorochrome, hapten) Fluorescence microscope Enzyme-couple chemiluminescence Autoradiography or phosphorimaging Enzyme-couple chromogenic

22. Nucleic Acid hybridization Southern blot DNA : DNA DNA fingerprinting and DNA typing DNA : DNA Colony hybridization DNA : DNA Northern blot RNA : cDNA Chromosomal DNA : DNA Microarray DNA: DNA cDNA : cDNA In situ hybridization (e.g fluorescence in situ hybridization; FISH)

23. Southern blots

25. RFLP (Restriction Fragment Length Polymorphisms)

26. DNA TestingbyAllele-Specific Cleavage

27. DNA TestingbyAllele-Specific Oligonucleotide hybridization

28. DNA fingerprinting

30. DNA typing

31. Northern blots (measuring gene activity)

32. FISH(Fluorescence in situHybridization) 22q11.12 Locating genes in chromosomes

33. Gene chips (Microarray)

34. Gene identification Southern blot FISH Immunoblots (Western Blots) DNA sequencing Restriction mapping

35. Restriction mapping (physical mapping)

36. Identification of a new gene Identification of the transcript mapping the start site and stop site measuring active transcripts Identification of the gene product quantitative and qualitative analysis Identification of the gene function gain of function loss of function Immunoblots (Western blots)

37. Mapping the start site of transcripts S1 mapping Primer extension Run-off transcription

38. S1 mapping the 5’ end

40. Primer extension

41. Run-off transcription

42. Mapping the stop site of transcripts S1 mapping

43. S1 mapping the 3’ end

44. Measuring active transcripts • Northern blot • In situ Histochemistry stain • Nuclear run-on transcription

45. Nuclear Run-on transcription

46. Immunoblots Immunoblots (also called Western blots) use a similar process to Southern blots • Electrophoresis of proteins • Blot the proteins from the gel to a membrane • Detect the protein using antibody or antiserum to the target protein • Labeled secondary antibody is used to bind the first antibody and increase the signal

47. Western Blots

48. Qualitative analysis of the cis/trans element activity Reporter gene activity Cellulose filter binding assay Gel mobility shift assay DNase footprinting DMS footprinting

49. Reporter gene