生物科技研究方法

1. 生物科技研究方法 Tools for analyzing gene expression Reference: fundamental molecular biology, Lizabeth Allison, 2007 蔡士彰

2. Central dogma DNA RNA protein

5. RNA DETECTION & ANALYSIS **Steady-state levels of RNA** ** RNA levels that exist as a result of ** Transcription (+) versus degradation (-) • METHODS: • Northern blotting • RNaseprotection assay • RT-PCR • Microarrays

6. RPA Add RNase RNA duplex protected

7. RT-PCR mRNA Reverse transcription PCR

8. Genome-wide comparison of mRNA expression between tumor and normal cells

9. Microarrays Massively parallel analysis of gene expression • screen an entire genome at once • find not only individual genes that differ, but groups of genes that differ. • find relative expression level differences Effective for identify regulatory networks

10. Reporter Assay Transfect embryo or ES cells DNA transfection methods 1. Calcium phosphate-DNA Coprecipitation method 2. DEAE-Dextran 3. Electroporation 4. Micro injection 5. Liposome Analysis of expression pattern in whole organism Direct enzymatic activity assay of cell lysate

11. The major approaches of DNA delivery Physical delivery Chemical delivery Viral vectors Bactofection (bacterial vectors)

12. Chemical delivery 1. Calcium-phosphate mediated transfection 2. polyplexes: eg. DEAE-dextran 3. Cationic liposomes: eg. Lipofectin, lipofectamine, lipofectase….

13. Physical delivery: electroporation, microinjection, gene gun (microballistics) Electroporation: Microinjection:

14. Reporter genes

15. X-gal IPTG: an inducer of β-galactosidase. This compound is used as a molecular mimic of allolactose Lactose is a disaccharide that consists of β-D-__________ and β-D-____________ molecules bonded through a β1-4 glycosidic linkage.

16. X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. 5-bromo-4-chloro-3-hydroxyindole then is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product.

17. Luciferase is a generic name for enzymes commonly used in nature for bioluminescence. The name itself is derived from Lucifer, which means light-bearer. The most famous one is firefly luciferase from the firefly Photinus pyralis. In luminescent reactions, light is produced by the oxidation of a luciferin (a pigment), sometimes involving Adenosine triphosphate (ATP). The rates of this reaction between luciferin and oxygen are extremely slow until they are catalyzed by luciferase The reaction takes place in two steps: luciferin + ATP → luciferyl adenylate + PPi luciferyl adenylate + O2 → oxyluciferin + AMP + light The reaction is very energy efficient: nearly all of the energy input into the reaction is transformed into light. As a comparison, the incandescent light bulb loses about 90% of its energy to heat.

18. luciferase (luc) systems firefly species Photinus pyralis Expressed luciferase catalyses oxidation of compounds called luciferans ( ATP-dependent process) These compounds emit fluorescense luminometer measurement Mice are injected with LUC+ salmonellas. Sensitive digital cameras allow non-invasive detection. For GT vectors pictures look the same

19. Chloramphenicol acetyltransferase is a bacterial enzyme that detoxifies the antibioticchloramphenicol. It is responsible for chloramphenicol resistance in bacteria. This enzyme covalently attaches an acetyl group from acetyl-CoA to the chloramphenicol molecule so that it is unable to bind to the ribosome.

20. Reporter gene systems chloramphenicol acetyl transferase (CAT) CAT is a bacterial enzyme that catalyzes the transfer of acetyl groups from acetyl-coenzyme A to the antibiotic chloramphenicol. (chloramphenicol deactivation) thin-layer chromatographic sheet Chloramphenicol is radiolabelled

21. GFP is an extremely stable protein of 238 amino acids with unique post-translationally created and covalently-attached chromophore from oxidised residues 65-67, Ser-Tyr-Gly Green fluorescent protein (GFP) autofluorescent protein from Pacific Northwest jellyfish Aequorea victoria ultraviolet light causes GFP to autofluoresce In a bright green color Jellyfish do nothing with UV, The activate GFP by aequorin (Ca++ activated, biolumuniscent helper)

23. GFP expression is harmlessfor cells and animals GFP construct could be used for construct tracking in living organism GFP labelled image of a human tumor. Vessel on the tumor surface are visible in black

28. Electrophoretic-Mobility Shift Assay (EMSA) DNA binding site 20-30 nt probe + DNA binding protein Protein/DNA complex

29. Electrophoretic-Mobility Shift Assay Incubate protein and DNA probe Load onto non-denaturing PAGE Resolve complexes & free probe protein concentration 0 - shifted probe free probe +

30. EMSA Resolution of multiple protein/DNA complexes: Proteins sharing a DNA binding element

31. G A B A G 0 0 *

32. DNAase I Footprinting G A G 0 0 100-300 bp DNA fragment with unique end-label Incubate with test protein(s) Digest with DNase I Run digested DNA on PAGE

33. Summary Methods to analyze transcription - detection of mRNAs - determination of transcriptional start sites -genome wide expression analysis by microarrays -reporter assay systems Methods to determine core promoter and/or enhancer elements -promoter mutagenesis -sequence alignment Methods to study DNA-protein interactions -EMSA, -DNase foot printing - Chromatin IP