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

Taq Polymerase• Thermus aquaticus DNA

polymerase• thermophilic organism• enzymes resistant to high

temperatures• 72-74o optimum

PCR Requirements• heat-stable DNA

polymerase• thermocycler• target DNA and

primers

STEP TEMP TIME NOTES

Denature 94-96o 0.5-2 min longer: denaturation, but enzyme, template

Annealing 15-25o < Tm 0.5-2 min higher/shorter: specificity, but yield

Extension 72-75o ~1 min (<kb) Taq processivity = 150 nucleotides/sec

•mix DNA, primers, dNTPs, Taq, buffer, Mg2+

• program thermocycler for times and temps –denaturation –annealing –extension

• 20-30 cycles• analyze amplified DNA

(amplicons)

PCR Protocol

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• 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