1. Gel
electrophoresis
analysis
Automated DNA analyzer

2. PCR Round 1 target DNA Double-stranded DNA Denaturation5' 3'
Double-stranded DNA
Denaturation 5' 3'
Primer annealing 5' 3' 3' 5'
Extension
Extension 3' 5'
repeat PCR cycles
DNA polymerase always adds nucleotides to the 3’ end of the primer

3. 3' 5'
PCR Round 2 5' 3'
After the second round of PCR, the number of long strands increases arithmetically and the number of short strands increases exponentially (the number of chromosomal strands is always the same).
denaturation 3' 5'
primer annealing 3' 5'
Short strand
extension
Long strand
Chromosomal strand

4. Temperature control in a PCR thermocycler
94 0C - denaturation
50 – 70 0C - primer annealing
72 0C - primer extension
94 0C - denaturation

5. [DNA] # PCR cycles After 25 cycles have 3.4 x 107 times more DNA
plateau is reached after
25-30 cycles
# PCR cycles

6. Multiplex PCR
Use of multiple sets of primers to detect more than one organism or to detect multiple genes in one organism. Remember, the PCR reaction is inherently biased depending on the G+C content of the target and primer DNA. So performing multiplex PCR can be tricky. or

7. Labelling approaches Real-Time PCR CYBR green TAQ-man probes
This technique allows quantitation of DNA and RNA. Reactions are characterized by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.
TAQ-man probes
FRET probes