Polymerase Chain Reaction

Polymerase Chain Reaction: 

Polymerase Chain Reaction -Varun C N

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Varun C N 2 Declaration All the materials that is shown in the slide has been taken from various sources (Which has been sited as appropriate) . The Non cited materials have been taken from public domains. None of them involves any of my work and hence I don’t claim any intellectual property rights over any of them. The material has been prepared only for educational purpose and I don’t take any personal responsibility for the matter produced. I have however taken maximum care to make data as reliable as possible Varun C N Feb 2012

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From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ 3 Varun C N- Polymerase Chain Reaction

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Mullis discovered the PCR procedure, for which he was awarded the Nobel prize in 1993 Kary Mullis accepting the Nobel Prize Awarded the Nobel Prize in Physiology or Medicine for his excellent work on the interpretation of the genetic code and its function in protein synthesis Vs 4 Varun C N- Polymerase Chain Reaction

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Varun C N- Polymerase Chain Reaction 5 A PCR cycle consists of three steps Melting of DNA Hybridization of primers DNA synthesis or extension of primers

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Varun C N- Polymerase Chain Reaction 6 Taken from: http://users.ugent.be/~avierstr/principles/pcr.html

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Varun C N- Polymerase Chain Reaction 7 Melting of DNA The temperature required for the melting of DNA can be approximately calculated by using the formula Tm o C = 2(A/T) + 4(G/C). Tm= { Δ H / ( Δ S+ Rln (c)}-{273.15 + 16.6 log [Na+]} The template concentration is considered to be negligible compared with the primer concentration, so the formula for calculating Tm is simplified such that it does not contain a parameter for template concentration.

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Varun C N- Polymerase Chain Reaction 8

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Varun C N- Polymerase Chain Reaction 9 Absorbance at 260nm Temperature AT rich GC rich

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Varun C N- Polymerase Chain Reaction 10 Isolated from human blood were analyzed using the RealHelix™ qRT-PCR Kit or Rotor-Gene SYBR Green RT-PCR Kit, and a primer set specific Human β- globin gene . Nanohelix, RealHelix™ qRT-PCR kit Melt Curve Analysis

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Varun C N- Polymerase Chain Reaction 11 Hybridization of primers Also known as Annealing of primers The solution is cooled to 54-65ºC to allow each primer to bind to a DNA strand. One primer hybridizes to the 3’end of the target on one strand, and the other primer hybridizes to the 3’ end on the complementary target strand. Two primers- Forward primer and the Reverse primers are used.

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Varun C N- Polymerase Chain Reaction 12 DNA synthesis or extension of primers The solution is heated to 72-75ºC, the optimal temperature for Taq DNA polymerase. The polymerase elongates both primers in the direction of the target sequence because DNA synthesis is in the 5’-to-3’ direction

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Varun C N- Polymerase Chain Reaction 13 http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2010/Streb/Orthologs.html

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Varun C N- Polymerase Chain Reaction 14 Fig: PCR phases in log view

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Varun C N- Polymerase Chain Reaction 15 For the optimization of PCR the following factors has to be considered 1. Quality and concentration of DNA template 2. Design and concentration of primers; 3. Concentration of magnesium ions 4. Concentration of the four deoxynucleotides (dNTPs) 5. PCR buffer systems 6. Selection and concentration of DNA polymerase; 7. PCR thermal cycling conditions 8. Addition and concentrations of PCR additives/co solvents 9. Use of the “hot start” technique

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Varun C N- Polymerase Chain Reaction 16 To measure the success of PCR amplification, 5 to 10 μL of the final PCR product is run on a 1 or 2% agarose gel and visualized by staining with Ethidium bromide. Taken from- Nature.com

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Varun C N- Polymerase Chain Reaction 17 More than a 30 modifications of the original PCR is in current use. However, the commonly used modifications are Simple PCR Real time PCR Reverse transcriptase PCR Nested PCR

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Varun C N- Polymerase Chain Reaction 18 McBride and Caruthers method of primer synthesis http://www.exiqon.com/ls/Documents/Scientific/Oligonucleotide-synthesis.pdf For additional details http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1275

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Varun C N- Polymerase Chain Reaction 19 Thank you for your kind attention You can also follow me at my blog. Varuncnmicro.blogspot.com