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Chapter 5 Exploring genes Foundation Enzymology of proteins that alter Nucleic Acids -Hybridization of DNA with DNA (cDNA) or RNA -Efficient sequencing methods -Deliver DNA to host organism Basic Tools • Restriction enzymes; • Blotting techniques (Southern, DNA; Northern, RNA); • DNA sequencing; • Solid phase synthesis of nucleic acids; • The polymerase chain reaction (PCR). Restriction enzymes • Proteins; • Restriction endonucleases, recognize specific DNA sequence and cleave at specific DNA sequence (scalpels or scissors); • Discovered by Werner Arber and Hamilton Smith and first used by Daniel Nathans; • Usually recognize palindromic sequences. A man a plan a canal panama “amanap lanac a nalp a nam a” Sticky Sticky Blunt Sticky Sticky Restriction fragments can be separated by gel electrophoresis and visualized. Southern Blotting -DNA is denatured to single strands before being transfered DNA sequencing: Sanger dideoxy method DNA sequencing with fluorescent tags A complete genome: Haemophilus influenzae DNA Synthesis: automated solidphase method The Polymerase Chain Reaction (PCR) • • • • • • Kary Mullis, 1984; Thermal cycler; Taq DNA polymerase; dNTP; DNA template; Primers. Heat to 95°C for 15 s Heated to 72°C, optimum for Taq polymerase The first cycle in PCR No new reagents added PCR • • • • Strand separation (denaturation, 950C); Hybridization of primers (annealing; 550C); DNA synthesis (extension) (720C); 20 to 30 cycles (220 to 230 molecules) Can be used for detecting bacteria (tuberculosis) and viruses (HIV) rapidly. -Very important for forensics (blood or hair etc.)and legal medicine (paternity and immigration) DNA and Forensics Recombinant DNA Technology • Pioneered by Paul Berg, Herbert Boyer, and Stanley Cohen in 1970s; • Revolutionized all aspects of biology. Restriction enzymes and DNA ligase Vectors for DNA cloning Plasmids; λ phage DNA molecules that can replicate autonomously in an appropriate host organism. Specific genes can be cloned from digests of genomic DNA: genomic library Detection of DNA with a radiolabelled probe after transfer from master plate to nitrocellulose and lysis Introns Can be a problem for bacteria expression of eukaryotic proteins Formation of cDNA Synthesis of proinsulin by bacteria Proteins with new function can be created -Deletion of a piece of DNA -Substitution, single aa oligonucleotide-directed mutagenesis. Half the progeny will have the mutant and half will not -Cassette Mutagenesis (see next slide) Cassette Mutagenesis Analyzing gene expression in eukaryotes at the mRNA level Quantitative PCR (qPCR) Determining mRNA expression level in cells via cDNA Levels of Gene Expression (amount of mRNA) Test all mRNAs at once Fluorescently labeled cDNA is bound to microarrays of DNA Microarrays prepared to complementary to known sequences obtained from genomic sequencing Genes inserted into Eukaryotic cells can be efficiently expressed. -Many post translational modifications do not occur in bacteria, thus eukaryotic genes are not always correctly expressed. -Provides insight into how genes are organized and expressed. -Methods for gene introduction include DNA precipitation by calcium phosphate followed by uptake (low efficiency), microinjection, or viruses Microinjection of DNA Transgeneic mice (growth hormone) Gene disruption by homologous recombination (knockouts) Consequences of gene disruption: sections of muscle from Normal (A) and gene-disrupted (B) mice, as viewed under Light microscope. Muscles do not develop properly in mice Having both myogenin genes disrupted. Double stranded RNA can inhibit protein expression