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:
1. Denaturation
2. Anneal
3. Extension
What is a primer?
• 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.
oligonucleotide
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 nonspecific 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
N
55-65oC
Y
GC content
45-55%
Y
Hairpin and selfdimerization
List of
acceptable
primers
N
Y
N
N
Cross Dimerization
Y
Excluded primers
Tm:
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