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Introduction to PCR.pptx

  1. An introduction to Central Dogma of Molecular Biology and Polymerase Chain Reaction XX
  2. Structure of DNA Source -
  3. Rosalind Franklin - Best known for her contributions to the discovery of the molecular structure of deoxyribonucleic acid (DNA) ( James Watson and Francis Crick with a model of DNA Helix ( their-dna-model)
  4. David Baltimore – Discovered reverse Transcriptase ( Stanley Prusiner – Discovered Prions ( -appointed-board-foundation-food-and-agricultural- research)
  5. Central Dogma of Molecular Biology • It was proposed by Francis Crick in 1957 – first published in 1958 • It states that genetic information flows in one direction - Deoxy-Ribonucleic Acid (DNA) is converted to Ribonucleic Acid (RNA) by Transcription, which is converted to Protein by Translation. • Most pathogens (including DNA Viruses) follow the Central Dogma, while RNA Viruses and Retroviruses follow the Modified Central Dogma Fig. Modified Central Dogma ( CenDog.html)
  6. Diagrammatic Representation of Central Dogma (
  7. Fig. Transcription and Translation (
  8. Equipment used in PCR Freezer (-20 °C or -80 °C) • Used for long term storage of samples • Equipped with adjustable shelves to accommodate various types of sample containers • Also has programmable thermostat and temperature monitoring systems Refrigerated Centrifuge • High speed centrifuge • Used for nucleic acid extraction Class II A2 Biological safety cabinet • Used in sample processing • Provides personnel, product and environmental protection through filtered, laminar and unidirectional airflow • Air is filtered through High Efficiency Particulate Air (HEPA) Filter, and cabinet is maintained at negative pressure PCR Workstation • Workspace enclosed on three sides, provides the space for preparation of master mix
  9. Thermal Cycler • Used to amplify segments of DNA via the Polymerase Chain Reaction • Thermal block - tubes holding the reaction mixtures can be inserted • Cycler - raises and lowers the temperature of the block in pre-programmed steps Spectrophotometer • Analytical instrument used for the objective calculation of visible light, UV light, or infrared light emission or reflection • Used to verify the purity of extracted DNA sample • Absorbance Ratio between 260nm/280nm is used for this purpose Real time PCR Machine • Thermal cycler equipped with an optical detection module to measure the fluorescence signal
  10. PCR Workstation Thermal Cycler
  11. Figure showing principle of Spectrophotometer ( Spectrophotometer (
  12. Real Time PCR Machines
  13. Principle of PCR  Overview • Polymerase Chain Reaction is a laboratory technique used for the production of a large number of copies DNA segment • Developed by Kary Mullis in the 1980s • Principle – Based on the ability of DNA polymerase enzyme to synthesize a new strand of DNA complementary to the offered template strand  A nucleotide can only be added to a pre-existing 3’-OH group, thus a primer to which it can attach is needed – permits the delineation of a specific sequence to be amplified  Components of PCR • DNA template : Sample DNA with target sequence : Exposed to high temperature to separate strands • Thermostable DNA Polymerase enzyme : Can be obtained from Thermus aquaticus (Taq DNA Polymerase) or Pyrococcus furiosus (Pfu DNA Polymerase) Heat resistant enzymes, and can generate new strands using primers • Primer : short single stranded nucleotide sequence, required for initiating the reaction - new DNA nucleotides are synthesized from ends of primers
  14. • Deoxy-nucleotidetriphosphates : Used for the synthesis of the new DNA strand • Buffer solution : maintains chemical environment for reaction process • Bivalent/Monovalent Cations : Either Mg2+ , Mn2+ , K+ or could be used  The reaction is carried out in Small Reaction Tubes (0.2-0.5 mL) in a Thermal Cycler (used to maintain high temperatures)  Process • Denaturation Reaction is heated to 94-98 °C for 20-30 seconds – causes denaturation of DNA (separation of DNA strands due to breakage of Hydrogen bonds between strands) • Annealing Reaction chamber is cooled to 50-65 °C for 20-40 seconds, to allow the attachment of primer to the separated DNA strands • Extension/Elongation Temperature is maintained at 72-75 °C - A new DNA strand complementary to template strand is created by adding free dNTPs in the 5'-to-3' direction
  15. Diagrammatic Representation of PCR Cycling (
  16.  The first 3 steps (Denaturation, Annealing, Extension) are considered as 1 cycle of the reaction – Number of copies of DNA produced = 2n (Here n is the number of cycles) Normally 30-40 cycles are performed • Final elongation The reaction temperature is kept at 70-74 °C for 5-15 minutes, it ensures that any remaining strands of ssDNA are fully elongated • Final Hold Reaction temperature is reduced to 4-15 °C. The obtained DNA samples may be stored at 4 °C for short periods, stored at -20 °C or - 80 °C for long term storage  Stages of PCR replication (PCR Amplification curve) 1. Exponential amplification - At every cycle, the amount of product is doubled (assuming 100% reaction efficiency) 2. Levelling off - The reaction slows as the DNA polymerase loses activity and as consumption of reagents, such as dNTPs and primers, causes them to become more limited 3. Plateau - No more product accumulates due to exhaustion of reagents and enzyme
  17.  Visualization • Agarose based Gel Electrophoresis – separates DNA fragments based on size (2% Agarose Gel is used) • Fluorescent based  Role of House Keeping Genes / Internal Control • They act as an Internal Control : Increase shows that PCR has been carried out normally • Positive IC result indicates that amplification has occurred and thus provides assurance that negative test results are truly negative • Increases sensitivity by identifying samples inhibitory to PCR Examples : Beta-Globin, Beta-Actin
  19. Variations of PCR • Since the technique was invented in the 1980s, PCR has been adapted and modified in many ways, giving rise to many variations • To date, there are 37 different variants of PCR used for both diagnostic and research purposes 1. Amplified fragment length polymorphism (AFLP) PCR 2. Allele-specific PCR 3. Alu PCR 4. Assembly PCR 5. Asymmetric PCR 6. COLD PCR 7. Colony PCR 8. Conventional PCR 9. Digital PCR (dPCR) 10. Fast-cycling PCR 11. High-fidelity PCR 12. High-Resolution Melt (HRM) PCR 13. Hot-start PCR 14. In situ PCR 15. Intersequence-specific (ISSR) PCR 16. Inverse PCR 17. LATE (linear after the exponential) PCR 18. Ligation-mediated PCR 19. Long-range PCR 20. Methylation-specific PCR (MSP) 21. Miniprimer PCR 22.Multiplex-PCR 23.Nanoparticle-Assisted PCR (nanoPCR) 24.Nested PCR 25.Overlap extension PCR 26.Real-Time PCR (quantitative PCR or qPCR) 27.Repetitive sequence-based PCR 28.Reverse-Transcriptase (RT-PCR) 29.Reverse-Transcriptase Real-Time PCR (RT-qPCR) 30.RNase H-dependent PCR (rhPCR) 31.Single cell PCR 32.Single Specific Primer-PCR (SSP-PCR) 33.Solid phase PCR 34.Suicide PCR 35.Thermal asymmetric interlaced PCR (TAIL-PCR) 36.Touch down (TD) PCR 37.Variable Number of Tandem Repeats (VNTR) PCR
  20. Other Variations of PCR Multiplex PCR • Involves amplifying multiple sequences in a single reaction – by using several pairs of primers annealing to different target sequences • Very useful for DNA fingerprinting, study of genetic mutations, microsatellites and SNPs Hot Start PCR • Optimizes the yield of amplified product and suppresses nonspecific amplification and formation of primer dimers • Done by – inactivating/inhibiting Taq Polymerase and late addition • Increases product yield • Provides higher specificity and sensitivity Reverse Transcriptase PCR (RT-PCR) • Sample RNA is converted to cDNA (complementary DNA), which is then subjected to PCR cycling and detection • Useful for detecting RNA viruses in a given sample • More efficient than Northern Blot method (used for RNA quantification)
  21. Diagram of Hot Start PCR (
  22. Real Time PCR  It is a laboratory technique used to monitor the amplification of DNA during the reaction, and not at the end  This is done by 2 methods 1. Adding non specific fluorescent dyes that bind to dsDNA 2. Sequence specific DNA probe with fluorescent reporter that is detected only after hybridization (FRET)  Non Specific Fluorescent Dyes • A DNA binding dye is added – binds to dsDNA, therefore increase in dsDNA (product) amount will cause increased fluorescence intensity  Fluorogenic Resonance Energy Transfer (Fluorescent reporter probe) • PCR is prepared, and reporter probe is added - both probe and primers anneal to the DNA target • Polymerization begins, and once polymerase reaches the probe, its 5'-3'-exonuclease degrades the probe, separating the fluorescent reporter • Fluorescence is detected and measured in a real-time PCR machine
  23. Example of Fluorescent probe - 5′-6-carboxyfluorescein-ACGTGGCACTGCGGCACGTGGT-6 (Used to detect Cytomegalovirus DNA)
  24. Nested PCR • Used to increase the specificity of DNA amplification - Two sets of primers are used in two successive reactions • One pair of primers generates DNA products • Products from 1st PCR are used as template in 2nd PCR – using two different primers whose binding sites are located (nested) within the 1st set • Minimizes non-specific products Semi-Nested PCR • Similar to Nested PCR, but 1 primer is common to forward and reverse steps Asymmetric PCR • Asymmetric PCR preferentially amplifies one strand of the target DNA - amplified linearly and no longer exponentially • Used in some sequencing methods and hybridization probing, to generate one DNA strand as product • Thermocycling done with a limiting amount of primers Quantitative PCR • Used to detect the number of copies of DNA / Pathogen Load • Two types 1. Absolute Quantitative PCR – Provides absolute measurement of starting copy number 2. Relative Quantitative PCR – Provides accurate discrimination between relative amounts of copy number
  25. Isothermal (Loop Mediated Isothermal Amplification) • Single-tube technique for the amplification of DNA • Target sequence is amplified at constant temperature (60–65 °C / 140-149 °F) using two or three sets of primers and a polymerase with high strand displacement activity • The amplification product can be detected by photometry or measuring the turbidity caused by magnesium pyrophosphate precipitate in solution (byproduct of amplification) • The reaction can be followed in real-time by measuring the turbidity or by fluorescence using intercalating dyes such as SYTO 9 • Advantages – simple, rugged, and low cost • Limitations - not useful for cloning
  26. References • • • • • • • • • • • • • • •
  27. Thank You