1. PCR Optimization
To optimize PCR the following must be evaluated and fine tuned
Target DNA
Too small or too large
Good or poor quality
Complete or incomplete
Primer selection
Specific or oligo (broad)
Polymerase
Taq
Pfu polymerase can proofread and repair DNA
Cycling Parameters
Annealing Temperatures
Extension temp
Phase time
Ex: taq polymerase can synthesize 1kb in 1 min
# of cycles to achieve the desired results
Mg2+ ion concentration
Based on experimentation

2. Techniques based on PCR
DNA Microarrays or DNA chips
A solid surface is printed with thousands of pieces of DNA each representing a gene sequence
Each spot consists of PCR amplified DNA
Specialized microarray printers print the matrix much like an ink-jet printer
mRNA from a host cell goes through RT-PCR to produce cDNA that has a fluorescent probe attached
The cDNA becomes the DNA probe for the printed microarray
A different fluorescent color can be added to another cell for comparison
Red cancerous cell
Green non-cancerous cell

3. DNA Sequencing PCR is performed using both dNTPs & ddNTPs
Dideoxy nucleoside triphosphates
The ddNTP is missing the OH at the 3’ site of ribose terminating the growing DNA chain
Each nucleotide is given a different fluorescent marker so that the ending nucleotides are easily identified
The sequence is read by a sequencing machine

4. PCR in Medicine Diagnosis Bacterial infections Viral infections
DNA barcoding of bacteria
Performed with real time PCR
Viral infections
RT-PCR
Uses reverse transcriptase to produce cDNA to be amplified