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PCR types and Trouble shooting
Ameer Effat M. Elfarash Dept. of Genetics Fac. of Agriculture, Assiut Univ.
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PCR types Inverse PCR Overlapping PCR
Reverse Transcriptase PCR (RT-PCR) Real Time PCR (qRT-PCR) Colony PCR Nested PCR Multiplex PCR Long PCR Gradient PCR Hot Start PCR Inverse PCR Overlapping PCR DNA fingerprinting RAPD AFLP RFLP etc..
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Conventional PCR Template DNA salts (ions) DNA Polymerase dNTPs
Primers DNA Polymerase Template DNA DNA nucleotides, the building blocks for the new DNA Template DNA, the DNA sequence that you want to amplify Primers, single-stranded DNAs between 18 and 30 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA
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Reverse Transcriptase PCR (RT-PCR)
RT-PCR is used to qualitatively detect gene expression
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Real Time PCR (qRT-PCR)
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Colony PCR
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Nested PCR
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Multiplex PCR
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Gradient PCR
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Hot Start PCR hotstart normal Used to optimize the yield of the desired amplified product in PCR and simultaneously to suppress nonspecific amplification.
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Alvin Submersible for Exploration of Deep Sea Vents
Long PCR 95ºC S 30-60 ºC S 72 ºC min Standerd PCR Long PCR 200 bp – 2K bp 5K -20K Alvin Submersible for Exploration of Deep Sea Vents
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Inverse PCR
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Overlapping PCR PCR 1 PCR 2 PCR 3
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SSCP ISSR AFLP SNP MASAP SAMPL DArT RFLP COS RAPD VNTR SRAP CAPS SCAR
RDA SAMPL Molecular Markers (DNA fingerprinting)
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Restriction Fragment Length Polymorphism
(RFLP)
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Restriction Fragment Length Polymorphism (RFLP)
Restriction site? Restriction Enzymes: E.g., EcoR1
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Randomly amplified polymorphic DNA
RAPD
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Amplified Fragment Length Polymorphism AFLP-PCR
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PCR Troubleshooting
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Experiment design Blank reaction Controls for contamination
Contains all reagents except DNA template Negative control reaction Controls for specificity of the amplification reaction Contains all reagents and a DNA template lacking the target sequence Positive control reaction Controls for sensitivity Contains all reagents and a known target-containing DNA template
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Experimental design 104 bp Negative Control Blank Reaction
Positive Control Patient 1 Patient 2 Patient 3 Patient 4 Molecular Marker 104 bp
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Avoiding Contamination
DNA sample preparation, reaction mixture assemblage should be performed in separate areas. A Laminar Flow Cabinet with a UV lamp is recommended for preparing the reaction mixture. New gloves should be used for DNA purification. The use of tips with filters for both DNA sample and reaction mixture preparation Autoclaving of all buffers is recommended.
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Troubleshooting PCR No PCR product - Marker + no product
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Troubleshoots Reagent PCR cycling PCR inhibitors Template DNA Primers dNTP’s Taq DNA Polymerase Buffer MgCl2 Thermal cycler. Detergent Phenol Heparin Heme Dyes (bromphenol blue) Urine
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Tissue type used for DNA extraction Quantity and quality of DNA
DNA template Tissue type used for DNA extraction Quantity and quality of DNA Length of the DNA fragment to be amplified
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Template DNA: Larger template DNA amounts usually increase the yield of non-specific PCR products.
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Too few Mg2+ ions result in a low yield of PCR product
MgCl2 concentration. Too few Mg2+ ions result in a low yield of PCR product Too many will increase the yield of non- specific products. MgCl2 (mM)
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Taq DNA polymerase. - Higher Taq polymerase concentrations than
needed may cause synthesis of non-specific products. No PCR product---Taq Expiered hotstart normal
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dNTPs. The concentration of 4 dNTPs (dATP, dCTP, dGTP, dTTP) should be equal in the reaction mixture.
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Primers. - The primer should not be self-complementary
or complementary to any other primer in the reaction mixture, to prevent primer- dimers and hairpin formation.
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Tm Too many bands – Low Tm Not optimized Well optimized
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Troubleshooting PCR Primer dimers and misprime:
Annealing temp. too low (dimers) or too high (misprime). excess primers Design primers carefully Size is the sum of two primer lengths. Reagent blank Molecular weight markers Misprime PCR product Primer dimers 5’ G 3’ C 3’ Primer Primer 2
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Cycle parameters
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PCR Inhibitors Ethanol Detergent Phenol Heparin Heme
Dyes (bromphenol blue) Urine High concentration of DNA
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What if you have too many bands !!!
Specificity of primers. Annealing temp too low. Contamination Primer Conc. Decrease cycles Decrease MgCl2 concentration Not optimized Well optimized
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