polymerase chain reaction presentation

7/31/2019 Polymerase Chain Reaction Presentation

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Polymerase chain reactionPolymerase chain reaction (PCR ) is a technique toamplify a single or few copies of a piece of DNA acrossseveral orders of magnitude, generating thousands tomillions of copies of a particular DNA sequence.


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The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reactionfor DNA melting and enzymatic replication of theDNA. Primers (short DNA fragments) containingsequences complementary to the target region along with a DNA polymerase (after which the method isnamed) are key components to enable selective and

repeated amplification.


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As PCR progresses, the DNA generated is itself used asa template for replication, setting in motion a chainreaction in which the DNA template is exponentially amplified. PCR can be extensively modified to performa wide array of genetic manipulations.


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PCR procedure

Different steps are processed during thermal cyclingprocess while using PCR.

Preparation of Reaction Mixture Reaction Mixture Set UpComponents of the Reaction Mixture Cycling steps


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Preparation of Reaction MixtureTo perform several parallel reactions, a reactionmixture is prepared which contains

bufferdNTPsprimersandTaq DNA Polymerase.


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they are taken in a single tube, which can then bealiquoted into individual tubes. MgCl2 and templateDNA solutions are then added. This method of settingreactions minimizes the possibility of pipetting errorsand saves time by reducing the number of reagenttransfers.


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Components of the Reaction Mixture Usually the amount of template DNA is in the range of 0.01-1 ng

Higher amounts of template DNA usually increase the yield of nonspecific PCR products


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Primers.primers are short dna sequences. During primerselection diffrent guide lines should be kept in mind.PCR primers are usually 15-30 nucleotides in length.Longer primers provide higher specificity The GC content should be 40-60%. The C and Gnucleotides should be distributed uniformly throughout of the primer..


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The primer should not be self-complementary orcomplementary to any other primer in the reactionmixture, in order to avoid primer-dimer and hairpinformation All possible sites of complementarity between primersand the template DNA should be noted.


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dNTPs.The concentration of each dNTP in the reaction mixture isusually 200 M. It is very important to have equalconcentrations of each dNTP (dATP, dCTP, dGTP, dTTP),

as inaccuracy in the concentration of even a single dNTPdramatically increases the misincorporation level. When maximum fidelity of the PCR process is crucial, thefinal dNTP concentration should be 10-50 M, since thefidelity of DNA synthesis is maximal in this concentrationrange. In addition, the concentration of MgCl2 should beselected empirically, starting from 1 mM and increasing in0.1 mM steps, until a sufficient yield of PCR product isobtained.


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Taq DNA Polymerase Usually 1-1.5 u of Taq DNA Polymerase are used in50 l of reaction mix. Higher Taq DNA Polymeraseconcentrations may cause synthesis of nonspecificproducts. However, if inhibitors are present in thereaction mix (e.g., if the template DNA used is nothighly purified), higher amounts of Taq DNA Polymerase (2-3 u) may be necessary to obtain a better yield of amplification products.


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OTHER THERMOSTABLE DNA

POLYMERASESStoffel fragment61kD fragment of Taq polymerase butapproximately two-times more thermostableand with optimal activity over a wider range of magnesium concentration.Recombinant Taq polymerase

This has the advantage over 'natural' Taq enzyme of greater batch conformity and,hence, higher reproducibility


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Cycling ConditionsThe complete denaturation of the DNA templateat the start of the PCR reaction is of key

importance. Incomplete denaturation of DNA results in the inefficient utilization of template inthe first amplification cycle and in a poor yield of PCR product. The initial denaturation should be

performed over an interval of 1-3 min at 95C if theGC content is 50% or less. This interval should beextended up to 10 min for GC-rich templates.


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If the initial denaturation is no longer than 3 min at95C, Taq DNA Polymerase can be added into theinitial reaction mixture. If longer initial denaturationor a higher temperature isnecessary, Taq DNA Polymerase should be added only after the initial denaturation, as the stability of theenzyme dramatically decreases at temperatures over95C.


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Denaturation Step Usually denaturation for 0.5-2 min at 94-95C issufficient, since the PCR product synthesized inthe first amplification cycle is significantly shorter

than the template DNA and is completely denatured under these conditions. If the amplifiedDNA has a very high GC content, denaturationtime may be increased up to 3-4 min. Alternatively,additives facilitating DNA denaturation - glycerol(up to 10-15vol.%), DMSO (up to 10%) orformamide (up to 5%) - should be used. In thepresence of such additives


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Primer Annealing Step Primer annealing at 55 oC to 65 oC for 20 seconds. However, if nonspecific PCR products are obtained

in addition to the expected product, theannealing temperature should be optimized by increasing it stepwise by 1-2C.


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Denature (heat to 95 oC)

Lower temperature to 56 oCAnneal with primers

Increase temperature to 72 oCDNA polymerase + dNTPs


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TYPES OF PCR AFLP PCR Allele-Specific PCR Assymetric PCR

Inverse PCR In Situ PCR Long PCR Nested PCR RT-PCR or Reverse Transcriptase PCR Real Time PCR Single Cell PCR Standard PCR


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Application of PCRIsolation of genomic DNA Amplification and quantification of DNA

PCR in diagnosis of diseases And many more


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Isolation of genomic DNAPCR allows isolation of DNA fragments fromgenomic DNA by selective amplification of aspecific region of DNA. This use of PCR ncludes,such as generating hybridization probes forSouthern or northern hybridization and DNA cloning, which require larger amounts of DNA,

representing a specific DNA region. PCR suppliesthese techniques with high amounts of pure DNA,enabling analysis of DNA samples even from very small amounts of starting material


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PCR 'fingerprints' methodsSome PCR 'fingerprints' methods have highdiscriminative power and can be used to identify genetic relationships between individuals, such asparent-child or between siblings, and are used inpaternity testing. This technique may also be used todetermine evolutionary relationships amongorganisms.


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DNA sequencingapplications of PCR include DNA sequencing todetermine unknown PCR-amplified sequences in which one of the amplification primers may be used inSanger sequencing method.

A lifi i d ifi i f
http://en.wikipedia.org/wiki/DNA_sequencinghttp://en.wikipedia.org/wiki/DNA_sequencing


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Amplification and quantification of DNA

Because PCR amplifies the regions of DNA that ittargets, PCR can be used to analyze extremely smallamounts of sample. This is often critical for forensicanalysis , when only a trace amount of DNA isavailable as evidence. PCR may also be used in theanalysis of ancient DNA that is tens of thousands of years old. These PCR-based techniques have beensuccessfully used on animals, such as a forty-thousand-year-old mammoth , and also on humanDNA, in applications ranging from the analysis of Egyptian mummies to the identification of a
http://en.wikipedia.org/wiki/Forensic_analysishttp://en.wikipedia.org/wiki/Forensic_analysishttp://en.wikipedia.org/wiki/ADNAhttp://en.wikipedia.org/wiki/Mammothhttp://en.wikipedia.org/wiki/Mummyhttp://en.wikipedia.org/wiki/Mummyhttp://en.wikipedia.org/wiki/Mammothhttp://en.wikipedia.org/wiki/ADNAhttp://en.wikipedia.org/wiki/Forensic_analysishttp://en.wikipedia.org/wiki/Forensic_analysis


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Quantitative PCR methods allow the estimation of theamount of a given sequence present in a sample atechnique often applied to quantitatively determinelevels of gene expression


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PCR in diagnosis of diseases

PCR allows early diagnosis of malignant diseases suchas leukemia and lymphomas , which is currently thehighest developed in cancer research and is already being used routinely.
http://en.wikipedia.org/wiki/Malignanthttp://en.wikipedia.org/wiki/Leukemiahttp://en.wikipedia.org/wiki/Lymphomahttp://en.wikipedia.org/wiki/Lymphomahttp://en.wikipedia.org/wiki/Leukemiahttp://en.wikipedia.org/wiki/Malignant


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PCR also permits identification of non-cultivatableor slow-growing microorganisms such asmycobacteria ,anaerobic bacteria , viruses .The basis for PCR diagnostic applications in

microbiology is the detection of infectious agentsand the discrimination of non-pathogenic frompathogenic strains by virtue of specific genes.
http://en.wikipedia.org/wiki/Mycobacteriumhttp://en.wikipedia.org/wiki/Anaerobic_organismhttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Anaerobic_organismhttp://en.wikipedia.org/wiki/Mycobacterium


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Thank you

polymerase chain reaction presentation

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