1 / 15

Python Programming on PCR Primers Design

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).

cuyler
Download Presentation

Python Programming on PCR Primers Design

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

  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 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

  15. 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

More Related