PCRPV92W11 - PCR & PV92 Polymerase Chain Reaction...

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Unformatted text preview: PCR & PV92 Polymerase Chain Reaction With PCR, any specific segment—the target sequence—within a DNA segment— sequence— sample can be copied many times (amplified) completely in vitro.! in All you need: • A heat-block that can rapidly and precisely change temperature (Thermocycler) • Primers bracketing the sequence of interest • A special heat-stable DNApolymerase from a bacteria inhabiting hot-springs • dNTPs • Buffer & cofactors for the polymerase • Source DNA as template Polymerase Chain Reaction 5¢ • The PCR procedure Target sequence 3¢ TECHNIQUE Each temperature cycle: 1. High heat (94°C) • “melt” DNA • dsDNAËssDNA 2. Low heat (45–60°C) • Allow primers to anneal to DNA 3. Medium heat (72°C) • Reduce nonspecific binding • Taq polymerase polymerizes new DNA on template DNA 4. Return to Step 1. • New DNA from Step 3 also used as template. Heyer 3¢ Genomic DNA 1 Denaturation: Heat briefly to separate DNA strands Cycle 1 yields 2 molecules 2 Annealing: Cool to allow primers to hydrogen-bond. 3 Extension: DNA polymerase adds nucleotides to the 3¢ end of each primer Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence 5¢ 5¢ 3¢ 3¢ 5¢ Primers New nucleotides PCR & PV92 Polymerase Chain Reaction (PCR) Types of DNA sequences in the human genome Exons (regions of genes coding for protein, rRNA, tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Alu elements (10%) Introns and regulatory sequences (24%) Repetitive DNA unrelated to transposable elements (about 15%) Simple sequence Large-segment DNA (3%) duplications (5–6%) Heyer Unique noncoding DNA (15%) Figure 19.14 PCR & PV92 PV92 locus on chromosome 16 Alu insertion sequence PV92 locus on chromosome 16 641 bp PCR product 941 bp PCR product Heyer ...
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This note was uploaded on 09/02/2011 for the course BIOL 6B taught by Professor Heyer during the Spring '10 term at DeAnza College.

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