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Polymerase Chain Reaction Polymerase Chain Reaction Presentation Transcript

  • Polymerase Chain Reaction By Sheetal Narkar
  • PCR Reaction Components
    • Template: previously isolated and purified.
    • Two primers: to flank the target sequence.
    • Four deoxynucleosides triphosphate (dNTPs): to provide energy and nucleosides for the synthesis of DNA.
    • Buffer system containing magnesium.
    • DNA polymerase
  • PCR Reaction Components
    • Template:
    • The recommended amount of template for standard PCR is:
    • 1-10 ng bacterial DNA.
    • 0.1-1 ng plasmid DNA.
    • Primers:
    • Primer concentration between 0.1 and 0.6  M are generally optimal.
    • Higher primer concentrations may promote mispriming and accumulation of non specific product.
    • Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yield of desired product.
    • Deoxynucleosides Triphosphate (dNTPs) Concentration:
    • Balanced solution of all four dNTPs minimize polymerase error rate.
    • Imbalanced dNTP mixture will reduce Taq DNA polymerase fidelity.
    • The final concentration of dNTPs should be 50-500  M (each dNTP).
    • They are usually included at conc. of 200  M for each.
    • Buffer system containing magnesium:
    • Providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
    • Generally, the Ph of the reaction buffer is (Ph 8.3 – 9.0) will give the optimal results.
    • .
    PCR Reaction Components
    • Mg+ Concentration,
    • The optimal MgCl2 concentration may vary from approximately 1mM-5mM, 1.5 mM is optimal in most cases.
    • DNA Polymerase:
    • The recommended amount is 0.5 – 2.5 units/50  l reaction.
    • Too little will limit the amount of product, while too much can produce unwanted non specific products and decreased specificity
    PCR Reaction Components
    • Initial Denaturation:
    • This step consists of heating the reaction to a temperature of 94-95°C which is held for 1-9 minutes.
    • Initial heating of the PCR mixture for 2 minutes at 94- 95C  is enough to completely denature complex genomic DNA.
    • Each cycle includes three successive steps: Each cycle takes as little as few minutes and it usually takes fewer than 20 cycles to produce as much amplified DNA as one needs.
    • Denaturation: One to several minutes at 94-96 C  , during which the DNA is denatured into single strands.
    Thermal Cycling Profile for Standard PCR
    • Annealing:
    • One to several minutes at 50-65 C  , during which the primers hybridize or "anneal" (by way of hydrogen bonds) to their complementary sequences on either side of the target sequence; and
    • Extention:
    • For fragments up to 3 kb primer extension is normally carried out at 72 C  , during which the polymerase binds and extends a complementary DNA strand from each primer and add approximately 60 bases per second at 72C  .
    • Post extension and holding :
    • Cycling should conclude with a final extension at 72 C  for 5 -15 minute to promote completion of partial extension products and then holding at 4 c  .
    Thermal Cycling Profile for Standard PCR
    • Number of Cycles:
    • The number of cycles required for optimum amplification varies depending on the amount of the starting material.
    • In optimal reaction, less than 10 template molecules can be amplified in less than 40 cycles to a product that is easily detectable on a gel stained with ethidium bromide.
    • Most PCR should , therefore, include only 25 – 35 cycles.
    • As cycle increases, nonspecific products can accumulate.
    • After 20- 40 cycles of heating and cooling build up over a million copies of original DNA molecules.
  • PCR Phases
    • Three phases:
    • Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise.
    • Linear: The reaction components are being consumed, the reaction is slowing, and products are starting to degrade.
    • Plateau: The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.
  • Variants of PCR
  • Reverse Transcriptase-PCR
  • Nested PCR
  • Hot Start PCR
    • Some components essential for polymerase activity is separated from the reaction mixture until the temperature in the tubes has exceeded the optimal primer annealing temperature usually 55-65 C ˚.
    • The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase.
    • Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step.
    • E.g. DNA Polymerase- Eubacterial type I DNA polymerase, Pfu. These thermophilic DNA polymerases show a very small polymerase activity at room temperature.
  • Quantitative PCR
    • Real Time PCR
    • Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring (Real-Time).
    • Real-time PCR uses a fluorescent reporter signal to measure the amount of amplicon as it is generated . This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles.
  • Colony PCR
    • Colony PCR is used for the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products.
    • Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE autoclaved dH2O in an microfuge tube.
    • Heat the mix in a boiling water bath (90-100C) for 2 minutes
    • Spin sample for 2 minutes high speed in centrifuge.
    • Transfer 20 μl of the supernatant into a new microfuge tube
    • Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.
  • Asymmetric PCR
    • Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other.
    • It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is ideal. 
    • PCR is carried out as usual, but with a great excess of one primers for the chosen strand.
  • AFLP PCR
  • AFLP PCR
    • Genomic DNA is digested with one or more restriction enzymes. tetracutter (MseI) and a hexacutter (EcoRI).
    • Ligation of linkers to all restriction fragments.
    • Pre-selective PCR is performed using primers which match the linkers and restriction site specific sequences.
    • Electrophoretic separation and amplicons on a gel matrix, followed by visualisation of the band pattern.
    AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA. AFLP can be also used for genotyping individuals for a large number of loci
  • Inverse PCR
    • Inverse PCR (Ochman et al., 1988) uses standard PCR (polymerase chain reaction)- primers oriented in the reverse direction of the usual orientation.
    • The template for the reverse primers is a restriction fragment that has been selfligated
    • Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA.
    • Applications
    • Amplification and identification of sequences flanking transposable elements, and the identification of genomic inserts.
  • Inverse PCR
  • Multiplex PCR
    • Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers.
  • In Situ PCR
    • In Situ PCR (ISH) is a polymerase chain reaction that actually takes place inside the cell on a slide. In situ PCR amplification can be performed on fixed tissue or cells.
    • Applies the methodology of hybridization of the nucleic acids.
    • Allows identification of cellular markers
    • Limited to detection of non-genomic material such as RNA, genes or genomes
  • In Situ PCR
  • Long PCR
    • Extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb).
    • Long PCR is useful only if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together.
    • Application- to clone large genes not possible with conventional PCR.
  • Allele-specific PCR
    • Used for identify of SNPs.
    • It requires prior knowledge of a DNA sequence, including differences between alleles.
    • Uses primers whose 3' ends encompass the SNP
    • PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer
    • Successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence
  • Polymerase Chain Reaction : Uses
    • The polymerase chain reaction (PCR) is a technique widely used in:
    • Molecular biology,
    • Microbiology,
    • Genetics,
    • Diagnostics clinical laboratories,
    • Forensic science,
    • Environmental science,
    • Hereditary studies,
    • Paternity testing, and
    • Many other applications………………………………………
  • Diagnosis of a variety of human disorders :
    • Infectious agents:
    • One area where the PCR technique will undoubtedly become a routine method, is the detection of infectious agents , such as pathogenic bacteria, viruses or protozoa.
    • Cancer:
    • Detection of malignant diseases by PCR.
    • Recurrence of hematological cancers has also been evaluated.
    • Detection of micro-metastasis in blood, lymph nodes and bone marrow.
    • Diagnosis of Genetic Diseases:
    • Single point mutations can be detected by modified PCR techniques such as the ligase chain reaction (LCR) and PCR-single-strand conformational polymorphisms (PCR-SSCP) analysis.
    • Detection of variation and mutation in genes using primers containing sequences that were not completely complementary to the template.
    • Prenatal Sexing and prenatal diagnosis of diseases :
    • Prenatal sexing is often required in families with inherited sex linked diseases. In these cases chorionic villus samples are ideal material for fetal sexing in the first trimester of pregnancy.
    • Prenatal Diagnosis of diseases
    • e.g. Prenatal diagnosis of many of the inborn errors of metabolism is possible by DNA markers.
    • Major role in the human genome project, replacing a single polymerase with a blend of a thermostable polymerase and a proofreading (Pwo DNA polymerase) made PCR an indispensable tool in the analysis and mapping of entire genomes by greatly extending the length of the sequence that could be amplified, increasing the amount of PCR product and providing higher fidelity during PCR.
    • Identify the level of expression of genes in extremely small samples of material, e.g. tissues or cells from the body by reverse transcription-PCR (RT-PCR).
    • Multiplex PCR , made it possible to compare two or more complex genomes, for instance to detect chromosomal imbalances.
    • Combining in situ hybridization with PCR made possible the localization of single nucleic acid sequences on one chromosome within an eukaryotic organism.
    • Amplification of archival and forensic material.
    • Identify testing for transplantation, HLA Typing.
    • PCR is used in research laboratories in DNA cloning procedures, Southern blotting, DNA sequencing, recombinant DNA technology.
    • Air Science USA       Applied Biosystems       Appropriate Technical Resources, Inc.       Azco Biotech, Inc.       Bio-Rad Life Science Group       Corbett Life Science       Eppendorf North America       Eppendorf North America       Finnzymes Inc.       GeneForge, Inc.       GENEWIZ Inc.       Kimberly-Clark Professional        
    LABREPCO       Laragen       MatriCal, Inc.       MIDSCI       Millipore Bioscience       Qiagen Inc.       Roche Applied Science       SciGene       Scimetrics, Inc.       Sigma-Aldrich Co. Labware       St. John Associates, Inc.       SuperArray Bioscience Corp.       Techne, Inc.       USB Corporation     PCR Manufacturers
  • Thank You