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  1. 1. by : Mahdi zarei M.Sc. Student ,clinical biochemistry Ferdowsi university of mashhad
  2. 2. History: • By 1971 researchers in Khorana's project, concerned over their yields of DNA, began looking at "repair synthesis" - an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene they are synthesizing. Although similar to PCR in using repeated applications of DNA polymerase, the process they usually describe employs just a single primer-template complex, and therefore would not lead to the exponential amplification seen in PCR. • Kary Mullis is generally credited with inventing PCR in 1983 while working for Cetus Corporation in Emeryville, California. While driving on Highway 128 from San Francisco to Mendocino, Mullis made an intellectual leap. He reasoned that by using two opposed primers, one complementary to the upper strand and the other to the lower, then performing multiple cycles of denaturation, annealing and polymerization he could exponentially amplify the piece of DNA between the primers.
  3. 3.  PCR was invented in 1983 by ( Kary mullis ) & he received the Nobel Prize in chemistry in 1993, for his invention.  It revolutionized biological methods specially in molecular cloning in a way that it has became an inseparable & irreplaceable part of molecular investigations. 3
  5. 5. Development….  PCR work was first published (1985)using Klenow polymerase unstable with heat New enzyme had to be added manually at each step Maximum length 400bp – not very practical  First reports using DNA polymerase from Thermus aquaticus (1988) • Taq-polymerase (Saiki et al, 1988) from Yellowstone National Park hot springs
  6. 6. Thermostable Polymerases Polymerase Taq pol Amplitaq (Stoffel fragment) Vent* T ½, 95oC 40 min Extension Type of Rate (nt/sec) ends 75 3’A 80 min >50 3’A 400 min >80 95% blunt Blunt Source T. aquaticus T. aquaticus Thermococcus litoralis Pfu >120 min 60 Pyrococcus furiosus Tth* 20 min >33 3’A T. (RT activity) thermophilus *Have proof-reading functions and can generate products over 30 kbp
  7. 7. Automation of PCR Developed automatic “thermocycler” programmable heat block… • The early PCR experiments , researchers had to rely on a series of water baths to maintain the different temperatures required by the procedure . “cycling” involved manual transfer of samples from one water bath to another at specified times. • In 1988, perkin-elmer introduced the thermal cycler , a revolutionary device that automatically and repetitively raised and lowered the temperature of the samples during PCR cycles . This allowed the PCR technique to be automated. Subsequent refinements of this device extended the flexibility and accuracy of pcr. • While in some old machines the block is submerged in an oil bath to control temperature, in modern PCR machines a Peltier element is commonly used.
  8. 8. PCR Thermocycler
  9. 9. Stage of pcr  Exponential amplification:  At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive.  Leveling off (linear)stage:  The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.  Plateau:  No more product accumulates due to exhaustion of reagents and enzyme.
  11. 11. Components of PCR Reaction • Template DNA • Flanking Primers • Thermo-stable polymerase • Taq Polymerase • dNTP • (dATP, dTTP, dCTP, dGTP) • PCR Buffer (mg++) • Thermocyler Thermus aquaticus
  12. 12. primers 1. PCR primers should be 10-24 nucleotides in length. 2. The GC content should be 40%-60%. 3. The primer should not be self-complementary or complementary to any other primer in the reaction mixture, to prevent primer-dimer and hairpin formation. 4. Melting temperatures of primer pairs should not differ by more than 5°C, so that the GC content and length must be chosen accordingly. 5. The melting and annealing temperatures of a primer are estimated as follows: if the primer is shorter than 20 nucleotides, the approximate melting temperature is calculated with the formula: Tm = 4(G + C) + 2 (A + T) 6. The annealing temperature should be about 5°C lower than the melting temperature.
  13. 13. PCR Buffer  Basic Components  20mM Tris-HCL pH 8.4  50mM KCl  1.5 mM MgCl2  Magnesium – Since Mg ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg2+ ions result in a low yield of PCR product, and too many increase the yield of non-specific products and promote mis incorporation.  Potential Additives  Helix Destabilisers - useful when target DNA is high G/CWith NAs of high (G+C) content.  dimethyl sulphoxide (DMSO),  dimethyl formamide (DMF),  urea  formamide  Long Targets >1kb. Formamide and glycerol  Low concentration of template: Polyethylene glycol (PEG)
  14. 14. Temperature  Denaturation  Trade off between denaturing DNA and not denaturing Taq Polymerase  Taq half-life 40min at 95 , 10min at 97.5  95  Annealing  Trade off between efficient annealling and specificity  2-5 below Tm  Extension  Temperature optimum for Taq Polymerase  72
  15. 15. Temperature 100 PCR Melting 94 oC 50 0 T i m e 3’ 5’ 5’ 3’
  16. 16. Temperature 100 PCR Melting 94 oC 50 0 T i m e 3’ 5’ Heat 5’ 3’
  17. 17. Temperature 100 Melting 94 oC 50 Melting o Extension 94 C Annealing 72 oC Primers 50 oC 0 T i m e 3’ 5’ 5’ 5’ 5’ 3’ PCR
  18. 18. Temperature 100 Melting 94 oC 50 Melting o Extension 94 C Annealing 72 oC Primers 50 oC 0 T i m e 3’ 5’ Heat 5’ 5’ Heat 5’ 5’ 3’ 30x PCR
  19. 19. Temperature 100 Melting 94 oC 50 Melting o Extension 94 C Annealing 72 oC Primers 50 oC 0 T i m e 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 30x PCR
  20. 20. Temperature 100 50 0 3’ 5’ 5’ Melting 94 oC Melting o Extension 94 C Annealing 72 oC Primers 50 oC T i m e 5’ 5’ 5’ 3’ Heat 5’ 5’ Heat 5’ 30x PCR
  21. 21. Temperature 100 50 0 3’ 5’ 5’ Melting 94 oC Melting o Extension 94 C Annealing 72 oC Primers 50 oC T i m e 5’ 5’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 30x PCR
  22. 22. Temperature 100 Melting 94 oC 50 0 3’ 5’ 5’ Melting o Extension 94 C Annealing 72 oC Primers 50 oC T i m e 5’ 5’ 5’ 5’ 3’ 5’ Fragments of defined length 5’ 5’ 5’ 5’ 5’ 30x PCR
  23. 23. More Cycles = More DNA Size Number of cycles Marker 0 10 15 20 25 30
  24. 24. Detection of pcr product Detection Visualization Agarose gel and/or polyacrylamide gel electrophoresis -EtBr staining (UV transilluminator, image analyzer) -Southern blotting (hybridization with labeled probe) -Silver staining Restriction endonuclease digestion -Agarose or polyacrylamide gel -HPLC Dot blots Hybridization with labeled probe High-pressure liquid chromatography UV detection Electrochemiluminescence Voltage-initiated chemical reaction/photon detection Direct sequencing Radioactive or fluoescent-based DNA sequencing
  25. 25. Controls for PCR  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
  26. 26. Interpretation of the PCR Results  The PCR product should be of the expected size.  No product should be present in the reagent blank.  Misprimes may occur due to non-specific hybridization of primers.(pcr product present in the negative control)  Primer dimers may occur due to hybridization of primers to each other.
  27. 27. Variations of the PCR
  28. 28. Hot Start PCR  It is a method for increasing specificity of PCR reactions.  This is a technique that reduces non-specific amplification during the initial set up stages of the PCR  The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase  DNA polymerase- eubacterial type I DNA polymerase, Pfu  Ampliwax or antibody are used in hot start pcr
  29. 29. Nested PCR  It is a method for increasing specificity of PCR reactions.  Two pairs (instead of one pair) of PCR primers are used to amplify a fragment.  First pair -amplify a fragment similar to a standard PCR. Second pair of primers- nested primers - bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one.  Advantage: Very low probability of nonspecific amplification
  30. 30. Touchdown PCR  It is a method for increasing specificity of PCR reactions.  Touchdown PCR uses a cycling program where the annealing temperature is gradually reduced (e.g. 1-2°C /every second cycle). The initial annealing temperature should be several degrees above the estimated Tm of the primers. The annealing temperature is then gradually decreased until it reaches the calculated annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing temperature.
  31. 31. 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.  Application : Amplification and identification of flanking sequences such as transposable elements, and the identification of genomic inserts.
  32. 32. Reverse Transcriptase PCR • Based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses. • First step of RT-PCR - "first strand reaction“-Synthesis of cDNA using oligo dT primers (37°C) 1 hr. • “Second strand reaction“-Digestion of cDNA:RNA hybrid (RNaseH)-Standard PCR with DNA oligo primers. • Allows the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.
  33. 33. 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.
  34. 34. Real-time PCR  real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (qPCR) or kinetic polymerase chain reaction.  Real-time PCR detects and measures the amplification target DNA as they are produced.  Unlike, conv. PCR, real-time PCR uses an oligonucleotide probe labeled with fluorescent dyes or an alternative chemistry, and a thermocycler equipped with the ability to measure fluorescence.
  35. 35. Real time PCR in comparison with other technical methods  amplification can be monitored real-time  no post-PCR processing of products (high throughput, low contamination risk)  Less time to getting results  No gel-based analysis at the end of the pcr reaction  Computer based analysis of the cycle-fluorescence time course  most specific, sensitive and reproducible  not much more expensive than conventional PCR (except equipment cost)
  36. 36. The Basic of Real time PCR
  37. 37. Two method for quantify real time pcr results: ABSOLUTE QUANTIFICATION & RELATIVE QUANTIFICATION
  38. 38. Absolute quantification
  39. 39. Relative quantification  This involves comparing the Ct values of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue. The Ct values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene(GAPDH ,rRNA ,…).  The comparative Ct method is also known as the 2-ΔΔCt method : ΔΔct=Δct sample – Δct reference Here, Δct,sample is the Ct value for any sample normalized to the endogenous housekeeping gene and ΔCt, reference is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.
  40. 40. Detection in real time PCR  Uses fluorescence as a reporter by Three general methods : 1. DNA-binding agents (SYBR Green)less accuracy. 2. Hydrolysis probes(TaqMan) 3. Hybridization probes (Light Cycler) most accurate & specific. Beacons, Scorpions
  41. 41. ® 1.SYBR    green DNA binding dye Binds to minor groove (dsDNA) Emits light when bound More double stranded DNA = more binding = more fluorescence  Forensically, can be used to calculate how much DNA was present before reaction.    Unspecific Melting curve analysis
  42. 42. The advantage of this technique is that it is relatively cheap as it can be used with any pair of primers for any target. However, as the presence of any dsDNA generates fluorescence, specificity of this assay is greatily decrease due to amplification of nonspescific PCR products and primer-dimers. Generating and comparing melting curves using the light cycler is one method of increasing the specificity of the reaction.
  43. 43. Melting curve analysis
  44. 44. Hydrolysis probe(taqman)
  45. 45. Dual hybridization probe
  46. 46. Molecular beacon
  47. 47. scorpions
  48. 48. references  MT Rahman , MS Uddin , R Sultana , A Moue , M Setu . Polymerase chean reaction (PCR) : A Short Rewiew . AKMMC J 2013: 4(1): 30-36  Manit A,Iqubal S ,Magali W ,Lyndon G et all .basic principles of real-time quantification pcr .mol.diagn.5(2),209-219.(2005)  http://www.nature.com/nprot/journal/v1  http://www.cryst.bbk.ac.uk/pps97/assignments/projects  http://www.invitrogen.com