1. Python Programming on PCR Primers Design
Ronny Chan
SoCalBSI
July 28, 2004

2. Polymerase Chain Reaction
PCR is a technique that is used to amplify a sample of DNA from miniscule amount of DNA (ex., DNA from a crime scene, archaeological samples, organisms that can’t be cultured).

3. Who developed PCR? PCR was developed by Kary Mullis.
Kary Mullis is a scientist and surfer from Newport Beach, California.
He won a Nobel Prize in Chemistry in 1993 for the development of PCR.
He was working for Cetus Corporation in the 70’s and received $10,000 bonus for the idea.

4. How is PCR used?
Medical Diagnosis: To detect and identify the causes of infectious diseases from bacteria and viruses.
Genetic testing: To determine whether a genetic mutation has been passed on (ex. cystic fibrosis).
Evolutionary study: To gather archaeological samples and analyzed for similarities/differences.
DNA fingerprinting: To profile DNA from blood, hair, and skin cells for criminal identification and forensics

5. Stages of PCR PCR is divided into 3 stages: Denaturation Anneal
Extension
The denaturation stage: the double-stranded DNA sample is separated into single strands by increasing the temperature to about 95 ºC.
The annealing stage: primers anneal to the DNA strands when the temp. is lowered to about ºC.
The extension stage: the DNA Taq polymerase duplicates the original DNA with the primers as guidelines.

6. What is a primer?
oligonucleotide
A primer is a short oligonucleotide which is the reverse complement of a region of a DNA template.
It would anneal to a DNA strand to facilitate the amplification of the targeted DNA sequence.

7. Primer Selection variables
Primer length
Melting Temperature
GC content
Hair-pin loop
Self-dimerization
Cross-dimerization

8. Primer Length Should be between 18 – 25 bases.
The longer the primer, the more inefficient the annealing.
If primers are too short, they will cause non-specific annealing and end up amplifying non-specific sequences.

9. Melting Temperature Formula (18-25 bp range):
Tm = 2(A+T) + 4(G+C)
The forward and reverse primers should be having similar Tm, or else amplification will be less efficient.
Melting Temperature should be between 55ºC and 65ºC.

10. GC Content GC% = (G + C) / length of seq * 100%
The base composition should be in the range of 45% to 55%.
Poly G’s or C’s can result in non-specific annealing.

11. Hairpin Loop Primers with hairpin loop may interfere
with annealing to the template by forming partially double-stranded structure.

12. Self-dimerization
Primers may form inter-primer homology with its own copies.

13. Cross Dimerization
Forward and Reverse primers may hybridize to form primer-dimer.

14. Algorithm for primer design
Input DNA sequence
Input the start and end of central region
Input the length of primers
Tm:
55-65oC N Y
GC content 45-55%
Excluded primers N Y
Hairpin and self-dimerization Y N
List of acceptable primers N
Cross Dimerization Y

15. References http://www.accessexcellence.org/AB/GG/polymerase.html