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Presentation On Polymerase Chain Reaction

Published in: Biology
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PCR is a type of method to test DNA or RNA of a species. This slides can help students understand more.

Jenn Y / Melaka

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  1. Chain Reaction
  2. Introduction Polymerase chain reaction (PCR) Developed by Kary Mullis in 1983 PCR is replacing immunological methods, in which Abs against a pathogen are used to identify the pathogen in a patient's blood. Abs are not detected until several days/weeks after the onset of infection
  3. Basic principles of PCR It serves to replicate and amplify target DNA Prepare master mix which contains: DNA template, DNA polymerase, nucleotides(A,T,C,G), primers It uses repeated cycles (3 steps/cycle) — 20 to 30 cycles: >Denaturation >Annealing/ Hybridization >Extension Heating master mix to 950C Separation of two complementary strands of dsDNA Cool down to lower temperature e.g., 550C Primers bind to the complimentary DNA Begin to attach additional complimentary nucleotides + Increase temperature to 720C (optimum for Taq polymerase) Addition of further nucleotides to the extending DNA strand Breaking of bonds that are not fully complimentary
  4. Novelty in Mullis's concept: "juxtaposition of two oligonucleotides, complementary to opposite strands of the DNA, to specifically amplify the region between them and to achieve this in a repetitive manner so that the product of one round of polymerase activity was added to the pool of template for the next round, hence the chain reaction" (Bartlett & Striling, 2003)
  5. DNA polymerase was originally extracted from the bacterium Escherichia coli. PCR reaction must be heated to denature the dsDNA Heating also irreversibly inactivated the E. coli DNA polymerase Fresh aliquots of enzyme had to be added at the start of each cycle Solution: DNA polymerase from Thermus aquaticus isolated from hot spring Heat stable Not rapidly inactivated at high temperature
  6. Initially, PCR technique was laborious and slow because one required manual transfer between water baths at different temperature Solution: thermocycling machine which replicates the temperature changes required for the PCR reaction without the need for manual transfer
  7. Preparation of PCR master mix: Basic components: Component sdH20 Forward primer I Reverse primer Buffer with Mgc12 dNTPs Taq polymerase DNA template [stock solution] [working solution] 10uM 10uM lox 10mM each 5 1-J/uL O.4uM O.4uM 0.2mM each 0.07 1-J/uL 50 ng/gL Volume per sample (gL) 8.79 0.6 0.6 1.5 0.3 0.21 3.0* *Adjust accordingly.
  8. PCR conditions 30 cycles 940C for 2 minutes (Initial denaturation) 940C for 30 seconds (Denaturation) 600C for 40 seconds (Annealing) 720C for 90 seconds (Extension) 720C for 7 minutes (Final extension)
  9. Gel casting Agarose gel dissolved in Tris borate/acetate EDTA (T BE or TAE) Concentrations: 0.8-2.0% Depending on the size of the DNA molecule Larger/longer DNA molecule -5 lower concentration Smaller/shorter DNA molecule -5 higher concentration
  10. Resolving gel Submerge agarose gel in buffer tank (TBE or TAE) Keep the DNA deprotonated Provides an ionic solution that allows current to pass through the water Load DNA sample (with loading dye) into the well Give colour and weight to DNA sample Make DNA molecules sink Allow visualization of progress of DNA migration white xylene cyanol cresol red bromophenol blue orange G
  11. Load marker (indicate sizes of DNA molecules) Turn on electric current DNA molecules are negatively charged due to all the phosphate groups in the backbones of DNA molecules Separation of DNA base on size Larger fragments move slower than smaller fragments Side view: O Sample loaded into well Gel Electric field and Direction of migration Negative (-) Electrode Plastic gel box Buffer Positive (+) Electrode
  12. Staining Stain with ethidium bromide (EtBr) for 10-15 minutes DNA in the gel is invisible to our naked eyes Visualize with UV light However, EtBr is a carcinogen & UV light damages eyes! Alternative: SYBR Green, Gel Red Aspect Sensitivity Mutagenicity Speed to diffuse into cells Compatibility with EtBr filter GelRed Non-mutagenic Does not diffuse into cells SYBR Green Non-mutagenic (diluted) Diffuses into cells rapidly No EtBr Highly mutagenic Can diffuse into cells
  13. Gel documentation View- control contrast & brightness Snap photos Save 1.5kbp
  14. If Troubleshooting little or no product is detected: 10-fold dilutions for template DNA Use more template Check for the presence of inhibitors in the template DNA Spiking the original PCR mix with dilutions of a known positive template Increase the temperature of the initial template denaturation step prior to cycling (standard: 950C for 5 min) Ensure template DNA is fully denatured (maximum number of priming sites) Vary concentrations of other buffer components (Taq polymerase, dNTPs, primers, Mg2+ pH) Add enhancers to the PCR mix Dimethylsulfoxide (DMSO), polyethylene glycol (PEG), glycerol, bovine serum albumin, betaine.
  15. If multiple products or a high- molecular weight smear is observed: Decrease the total number of cycles Increase annealing temperature Vary concentrations of other buffer components (Taq polymerase, dNTPs, primers, Mg2+ pH) Attempt band purification followed by reamplification
  16. 1. 2. 3. 4. 5. Refe re n s : Roux, K. H. (2009). Optimization and troubleshooting in PCR. Cold spring harbor protocols laboratory press, 4(4), 1-6. Joshi, M. & Deshpande, J. D. (2010). Polymerase chain reaction: methods, principles and application. International journal of biomedical research, 1(5), 81-97. Santos, C. F. D. , Sakai, V. T. , Machado, M. A. A. M. , Schippers, D. N. & Greene, A. S. (2004). Reverse transcription and polymerase chain reaction: principles and applications in dentistry. Journal of applied oral science, 12(1), 1-11. Goessl, C. et al. (2001). Detection of prostate cancer using methylation-specific PCR. QIAGEN news, 4, 5-8. Niemeyer, C. M. , Adler, M. & Wacker, R. (2005). Trends in biotechnology, 23(4), 208-216.