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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).

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python programming on pcr primers design

Python Programming on PCR Primers Design

Ronny Chan

SoCalBSI

July 28, 2004

polymerase chain reaction
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
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
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
Stages of PCR
  • PCR is divided into 3 stages:
  • Denaturation
  • Anneal
  • Extension
what is a primer
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 Selection variables
  • Primer length
  • Melting Temperature
  • GC content
  • Hair-pin loop
  • Self-dimerization
  • Cross-dimerization
primer length
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
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 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
Hairpin Loop
  • Primers with hairpin loop may interfere
  • with annealing to the template by forming partially double-stranded structure.
self dimerization
Self-dimerization
  • Primers may form inter-primer homology with its own copies.
cross dimerization
Cross Dimerization
  • Forward and Reverse primers may hybridize to form primer-dimer.
algorithm for primer design
Algorithm for primer design

Input the length of primers

Input DNA sequence

Input the start and end of central region

Tm:

55-65oC

N

Y

GC content 45-55%

N

Excluded primers

Y

Hairpin and self-dimerization

Y

N

List of acceptable primers

N

Y

Cross Dimerization

references
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