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Molecular Genetic Methods

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Laura Parker

Lecture 4 – Molecular Genetic Methods I September 11, 2006 Note: The notes for this and the following lecture will be terse; these are methods lectures, and Dr. Nathans has rather condensed notes with many helpful figures. Restriction enzymes make DS breaks in DNA at specific unmethylated palindromic sequences. Plasmids can hold several kb of info, contain an origin of replication, and a resistance gene 3’ of the origin so you can select for those bacteria that fully took up the plasmid. Libraries contain DNA segments in a vector (viz., a plasmid or a virus). Genomic libraries are made by chopping genomic DNA with a restriction enzyme and inserting them into phage genomes. cDNA libraries are made by reverse-transcribing pre-spliced mRNA and then inserting this DNA into a plasmid. Denaturation and renaturation of DNA is the key to priming PCR, to find related sequences (old school), hybrid probing via Southern blot (DNA) and Northern blot (RNA). Hybridization of labeled nucleic acid probes to a sequence of interest shows up as a signal on nitrocellulose paper. Detecting DNA from your probes is done via radioactive isotopes, enzymatic detection (crosslink an active enzyme to your sequence), or fluorescence (used for DNA chips). Synthetic DNA can be made 3’-5’ on resin beads by using labile blocking groups on the 5’ end of incoming nucleotides. Good for making PCR primers. Sanger sequencing, covering only the fluorescent method here, requires a 5’ primer and fluorescent ddNTPs. ddNTPs will be incorporated randomly, cutting your sequence off and colorinthall of the strands of X length (say all 55bp strands will be Green, indicating that the 55 nucleotide is adenosine). Shortest strands migrate to the bottom of the gel. PCR involves knowing the 5’ and 3’ terminal sequences so you can build DNA primers. It lets you amplify a sequence of DNA for cloning or forensics, perform site-directed mutagenesis, RT-PCR to quickly amplify cDNA from mRNA, or assemble complex arrangements of various DNA sequences. DNA chip hybridization benefits from high-resolution lithography designed for microchips. It involves making a chip with ~250,000 slots per cm, then using a photolabile mask and NTP washes to build up a different derivatized DNA sequence in each slot. Especially useful for comparing RNA cellular populations, one probe labeled with red-fluorophore-derivatized DNA and the other with green. Also useful for looking at gene copy number between normal and cancer cells. Also good for detecting which region of DNA a binding protein actually binds (involves cross-linking and purification). Lecture 5 – Molecular Genetic Methods II September 12, 2006 Western blotting is like Southern blotting for proteins. Instead of a probe-DNA to DNA interaction, there is a labeled-antibody to protein interaction. Detects presence of protein of interest. Protein tagging is helpful for producing and then purifying a protein from a known cDNA (or sequence). I like the hexahistidine tag; GST and epitope tagging are also used. Yeast two hybrid screening lets you see which other proteins bind to your protein of interest (hybridized to a binding-depe
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