Final_notes - Lec 5 - DNA Length ~2 meters (the thread and...

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Lec 5 - DNA Length ~2 meters (the thread and the tennis ball, packing); 23 sets of chromosomes; Same DNA in every cell; Double helix ~2 nanometers in diameter; Exons --- code for proteins; Base-pair, end to end ~1.1nm; RNA Polymerase reads DNA and produces a complementary RNA strand; DNA Polymerase reads DNA and produces a complementary DNA strand; nucleotide pairs 3.2x10^9; 30,000 genes; DNA has polarity/directionality 3’ end = hydroxl end where new bases get added 5’ phosphate end =Terminus of the strand; hydrogen bonding; G-C pairs most prevalent; melt 90-95, hybridize 40-60c; ELECTROPHORESIS – restriction enzymes cleave DNA to smaller pieces that are loaded into wells in porous gel; electric field PCR - Replication of a unique region of the genome to Identify an organism; Forensics; Gene replication; Dev. cDNA library; replic. to provide a detectable signal; 2^n process (n cycles) cDNA – library of DNA that only codes for genes (no introns) Lec 6 – PCR Amplicon -- ssDNA that is amplified via DNA Polyermase Exonuclease -- Enzyme that clips off any unpaired bases “proof reading”; Clips off bases that are Mismatched; Clips off enough nucleotides to prime addition of nucleotides by polymerase FRET = Förster resonance energy transfer (FRET) describes an energy transfer mechanism between two chromophores Primer -- short, single strand of RNA that locates the start and end of a replication region for PCR Probe -- piece of RNA or DNA labeled with Reporter that locates a specific region of DNA by hybridization Reporter -- A Radioactive or Fluorescent signal that is detectable Exogenous DNA -- DNA outside an organism Cycle -- One cycle of PCR replication PCR-- Polymerase Chain Reaction QPCR -- Quantitative PCR RT-PCR -- Reverse Transcriptase PCR real time-PCR -- Real Time PCR ICC-PCR -- Integrated Cell Culture PCR Taq polymerase – no exonuclease; Pfu and Vent both have exonuclease PRIMER DESIGN : Uniqueness: ensure correct priming site; Length: 17-28 bases. Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible; Optimize base pairing: it’s critical that the stability at 5’ end be high and the stability at 3’ end be relatively low to minimize false priming. Melting Tm between 55-80 ˜C are preferred; Assure that primers at a set have annealing Tm within 2 – 3 ˜C of each other. Minimize internal secondary structure: hairpins and dimmers shall be avoided. REPORTERS
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This note was uploaded on 04/24/2008 for the course ENGR 213 taught by Professor Clague during the Winter '08 term at Cal Poly.

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Final_notes - Lec 5 - DNA Length ~2 meters (the thread and...

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