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