Taq polymerase will read the template and add dNTPs in a 5 3 manner These three

Taq polymerase will read the template and add dntps

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Taq polymerase will read the template and add dNTPs in a 5-3 manner These three steps will be done several dozens times o Every cycle exponentially increases the number of replications Occurs in thermocycler o “touchdown”; annealing temperature varies and slowly decrease o 1 minute, 45 seconds, 2 minutes 3) Sensitivity Most important consideration: contaminated DNA is easily amplified Poor primer design o Must be designed to minimize self-annealing and creation of primer dimers 4) D1S80 Locus Short tandem repeat (STR) region on human chromosome 1 o 100-300 bp region that have undergone duplications or deletion over time (variable number tandem repeats) 16 bp repeat o GAGGA CCACC (G/A)G(C/A)AA G o If repeated 18 times, said to have “18” allele o 80% heterozygous and 20% homozygous Hetero: different number of repeats for each copy of chromosome 1 D1S80 primers flank locus and provide Taq polymerase with starting point to amplify locus Agarose gel will separate fragments (if homo = one band, hetero = two bands) 5) Bacterial Transformation Insert pLAF plasmid into bacteria o Make chemically competent : competent to take up foreign DNA o Success = transformed Visually select transformed cells o Transformants vs nontransformants o Use of selective marker and different plate environments Every “lump” = billions and billions of cells (colony) 6) Steps of Bacterial Transformation Make chemically competent Thaw chemically-competent and keep on ice Add foreign plasmid DNA on ice Incubate mix of cell with DNA on ice (attracts DNA to the membrane) Heat shock (allows DNA to go through the membrane and into the cell) Return on ice (reduces movement of DNA and promotes exogenous DNA entering)
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LB broth (promotes bacterial growth) Spread on different agar plats using spreader Incubate 7) pLAF Plasmid (again) selective marker: ampicillin resistance gene tyrosinase (codes for copper-containing pigment) lac promoter: in presence of lactose, tyrosinase gene is transcribed o cofactor (copper) and substrate (tyrosinase) must also be present relationship between genotype and phenotype 8) Application: Recombinant DNA techniques DNA fingerprinting and genetic analysis Medical Diagnosis Viral transmission events can be traced back through time among individuals o Sister-group relationship among lineages 9) Bioinformatics BLAST (Basic Local Alignment Search Tool) o Algorithm used to search database for regions of similarities Homologous Sequences o Orthologs: genes separate by speciation o Paralogs: genes produced by gene duplication within the same species 10)HIV Comes from eating primates (probably from at least three species) Entered between 1920s-1930s Global epidemic: rural/urban transition, sexual practices, travel, health practices, drug use, wars 11)Obtaining Genomic DNA from cheek cells Rinse with saline solution and into tube Pipet into microcentrifuge tube Centrifuge for 5 minutes Save cell pellet Resuspend cell pellet in residual rinsate Transfer to solution with chelex beads Water bath, flick
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