Transcript Chapter 5
Chapter 5 Molecular Tools for Studying Genes and Gene Activity _ + In neutral pH buffer Friction force logarithmic For the DNAs in the size ranges ~Mb Pre-stained protein markers Labeled Tracers Autoradiography b-emitters: 3H, 14C, 35S, 32P Radioactive DNA fragments Phosphorimaging Greater linearity than autoradiography Radioactive sample B-ray Molecules (ground state) in Image plate Excited molecules in image plate Energy released from excited molecules by a laser bean from phosphorimager Detected by computerized detector Liquid Scintillation Counting Non-radioactive Tracers Using Nucleic Acid Hybridization Southern Blots: Identifying Specific DNA Fragments (Edward Southern--the pioneer) Band number > 1 ??? DNA Fingerprinting and DNA Typing Alec Jeffreys (1985): minisatellite (repeated) sequence in a-globin gene also in the whole human genome Minisatellite sequence has no RE site for HaeIII Unrelated Caucasians Monozygotic twins Forensic Uses of DNA Fingerprinting and DNA Typing Northern Blots: Measuring Gene Activity Poly(A)+ RNA: from rat tissues Probe: G3PDH (glyceraldehyde-3-phosphate dehydrogenase) In situ Hybridization: Locating genes in chromosomes FISH: Fluorescence in situ hybridization DNA Sequencing 1975 Frederick Sanger Alan Maxam Walter Gilbert 3 billion bases of human genome The Sanger Chain-termination Sequencing Method Using didexoy nucleotides CAAAAAACGG-------- Automated DNA sequencing Maxam-Gilbert Sequencing (Dimethyl sulfate: guanine) (piperidine) Protein Engineering of Cloned Genes: Site-directed Mutagenesis Change of protein function by change of the gene sequence Step 1 G C T A G PCR G C Step 2 PCR C Step 3 G C PCR G C PCR with + Mapping and Quantifying Transcripts Mapping: locating the starting and stopping points of transcripts Quantifying: measuring how much of a transcript exists at a certain time S1 Mapping for 5’-end of a transcript 5’ 3’ S1 Mapping for 3’-end of a transcript 3’ 5’ Primer Extension: To map the end of a transcript with one-nucleotide accuracy S1 nuclease tends nibble a bit on the RNA-DNA hybrid, and A-T-rich regions can melt transiently TTCGACTGACAGT Run-off Transcription To check the efficiency and accuracy of in vitro transcription (can be used to assay the promoter before or after mutation) Run-off Transcription Measuring Transcription Rates In Vivo Nuclear Run-on Transcription Initiation of new RNA chains in isolated nuclei does not generally occur (using heparin to inhibit free RNA polymerase and prevent re-initiation) Reporter Gene Transcription CAT: Chloramphenicol (CAM) acetyl transferase CAM: Protein synthesis inhibitor acetylation by CAT Loss inhibitor activity Other reporter enzymes: b-galatosidase luciferase Assaying DNA-Protein Interactions Filter Binding Nitrocellulose membrane binds proteins and ssDNA but not dsDNA dsDNA-protien complex do bind to NC Gel Mobility Shift Assay (Electrophoresis Mobility Shift Assay, EMSA) DNase Footprinting protein DMS Footprinting Methylating agent: Dimethylsulfate * * 1,4 no protein added Knockouts Brown: dominant