1. 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)

2. Geometric Amplification20 21 22 2n
1st cycle
2nd cycle
nth cycle 

3. 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

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 ( 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

10. 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)

11. 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
bp (amplicon)
20-26 bases (probe)
Tm of probe 8-10o > annealing temperature

12. 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