Molecular Genetics Flashcards
Lac operon
Monod and Jacob
Genes can be switched off and on
Proof of inhibitor: found mutants that never turn off B-gal gene
Normal bacteria donates plasmid (conjugation) with functioning B-gal gene, immediately produces B-gal, wild-type must produce inhibitor
Inhibitor blocks mRNA transcription
Semi-conservative replication
Meselson and Stahl
E.coli grown in media containing 15N –> incorporated into their DNA
Cells were suddenly switched into 14N containing media
First generation cells had DNA 50% 15N and 50% 14N
Northern blotting
Labelled antisense DNA probe to identify mRNA
(mRNA separated by size on gel, blotted onto filter)
Probe hybridises to complementary mRNA - determine size and quantity of mRNA on sample
Southern Blotting
Template is instead restriction-enzyme-treated DNA
Probe hybridises to determine quantity of DNA in sample
Western blotting
Protein samples through denaturing polyacrylamide gel, transfer to filter
Filter probed with antibody
Antibody binding quantified using enzyme-conjugated secondary antibody and assayed colorimetrically
Sanger method
Random interruption to PCR by enzymatic cleavage
4 ddNTPs
Incorporated by DNA polymeranbut can’t be extended
dNTP and ddNTP added randomly at each location
Fluoresctly labelled, so can be visualised with a laser
Fluorescence in situ hybdrisation
Hybridise fluorescent DNA probe to denatured chromosomal DNA
See translocation, elongation, truncated, portions, pin point chrosmome location
Chrosmome painting
Ultra-FISH
Combination of multiple hybridisation form many loci spanning a whole chrosmome to visualise each one
Reverse transcriptase PCR
Reverse transcribes DNA from RNA templates, particularly as eukaryotic share the common 3’ end
Quicker as only transcribes exons
Electrophoresis
Separation of complex of solutes by virtue of electrical charge and size
Evidence for Poly(A) tail
Block synthesis of tail with 3’ deoxyadenosine
mRNA could still be transported out the cell but was much less effective template for protein synthesis
Spliceosome structural organisation
Electron microscopy - characterise snRNPs
X-ray crystallography and NMR - individual snRNPs or spliceosomal proteins
Biochemical probes - spatial organisation
Fluorescent probes show protein-protein and RNA-RNA interaction
