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Blotting Techniques: Limitations needs g amounts of DNA DNA needs to be relatively pure

Polymerase Chain Reaction (PCR). specific DNA fragment(s) are enzymatically amplified 10 6 -fold amplification possible can detect single molecule tolerates impure DNA assay time < day. Blotting Techniques: Limitations needs g amounts of DNA DNA needs to be relatively pure

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Blotting Techniques: Limitations needs g amounts of DNA DNA needs to be relatively pure

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  1. Polymerase Chain Reaction (PCR) • specific DNA fragment(s) are enzymatically amplified • 106-fold amplification possible • can detect single molecule • tolerates impure DNA • assay time < day • Blotting Techniques: Limitations • needs g amounts of DNA • DNA needs to be relatively pure • assay time: several days to > week

  2. Geometric Amplification 20 1st cycle  21 2nd cycle  22 nth cycle   2n

  3. PCR Requirements • heat-stable DNA polymerase • thermocycler • target DNA and primers • Taq Polymerase • Thermus aquaticus DNA polymerase • thermophilic organism • enzymes resistant to high temperatures • 72-74o optimum

  4. PCR Protocol • mix DNA, primers, dNTPs, Taq, buffer, Mg2+ • program thermocycler for times and temps • denaturation • annealing • extension • 20-30 cycles • analyze amplified DNA (amplicons)

  5. Design of Oligonucleotide Primers • analyze sequence with computer • amplicon length (250-1000 bp) • uniqueness (18-28 bases) • Tm > 55o • 50% GC composition • 3'-GC 'caps' • no internal complementarity • no 'primer dimers' • HPLC purification (optional)

  6. RNA-PCR • aka RT-PCR • make a complementary copy of mRNA • use the cDNA in PCR reaction • 3 basic strategies

  7. Quantitative PCR • titrate with known amounts of ‘competitor’ • laborious • ‘real time’ PCR • use fluorescent tags and ‘light cycler’ • dsDNA binding dye (eg., SYBR green) • specific ssDNA probes • measure accumulation of product during the PCR reaction

  8. Specific RT-PCR Probes • DNA-intercalating dyes are non-specific • accumulation of spurious amplicons • primer dimers and target DNA • no multiplexing • ssDNA probes against amplicon add specificity • detection based upon fluorochrome and quencher pairs • hydrolysis probes (aka Taqman or F-Q probes) • molecular beacons (aka hairpin probes)

  9. Taqman Probes • probe contains fluorescent tag and quencher • exonuclease activity of Taq polymerase releases fluorescent tag • fluorescence  each cycle • high background from probe • Primer/Probe Design • 50-150 bp (amplicon) • 20-26 bases (probe) • Tm of probe 8-10o > annealing temperature

  10. Problems and Limitations • minor DNA contamination can be a serious problem • need to know flanking sequences to design primers • Precautions • gloves • filtered pipette tips • sterile hood • decontaminate (eg, UV) • aliquot reagents • add target DNA last • no target DNA control • prepare ‘+’ control elsewhere • Unknown Flanking • inverse PCR • add ‘anchors’ • use random primers • RAPD • AFLP

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