Polymerase Chain Reaction (PCR)

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

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

heat-stable DNA polymerase thermocycler target DNA and primers 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

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)

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)

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

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

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)

Taqman Probes Primer/Probe Design 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

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