Python Programming on PCR Primers Design Ronny Chan SoCalBSI July 28, 2004
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).
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.
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
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 50-65 ºC. The extension stage: the DNA Taq polymerase duplicates the original DNA with the primers as guidelines.
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.
Primer Selection variables Primer length Melting Temperature GC content Hair-pin loop Self-dimerization Cross-dimerization
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.
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.
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.
Hairpin Loop Primers with hairpin loop may interfere with annealing to the template by forming partially double-stranded structure.
Self-dimerization Primers may form inter-primer homology with its own copies.
Cross Dimerization Forward and Reverse primers may hybridize to form primer-dimer.
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
References http://www.accessexcellence.org/AB/GG/polymerase.html http://www.accessexcellence.org/AB/BC/Kary_B_Mullis.html http://bioweb.uwlax.edu/GenWeb/Molecular/Seq_Anal/Primer_Design/primer_design.htm http://www.karymullis.com/ http://www.bioteach.ubc.ca/MolecularBiology/PolymeraseChainReaction/ http://www.emblheidelberg.de/ExternalInfo/geerlof/draft_frames/flowchart/clo_pcr_strategy/primer_design.html http://www.ncbi.nlm.nih.gov/Class/NAWBIS/Modules/DNA/dna9.html http://www.dnalc.org/shockwave/pcranwhole.html http://www.alumni.ca/~leema3m/bg/pcr.html http://marvin.ibest.uidaho.edu/~heckendo/CS504/Students/P/waltari.pdf