1. POLYMERASE CHAIN REACTION Chapter no: 9 Mohammad Hosein Bakhshi

2. WHO INVENTED IT ? Dr. Kary Banks Mullis born on December 28, 1944 Ph.D. degree in biochemistry from the University of California, Berkeley 1983: Dr. Kary Mullis developed PCR 1993:Dr. Kary Mullis shares Nobel Prize in Chemistry for conceiving PCR technology. 3. WHAT IS THAT? the automated process that allows a specific DNA chain also called primers to be replicated over and over again to produce more copies of that DNA. This machines purpose is to alter the temperature so that the primers can be heated and cooled to perform the processes correctly Thermo cycler 4. PURPOSE OF PCR To amplify a lot of double-stranded DNA molecules (fragments) with same (identical) size and sequence by enzymatic method and cycling condition. In vivo (Cloning) In vitro (PCR) DNA amplification 5. ADVANTAGES Much faster than using vectors Only a little bit of target DNA is needed DISADVANTAGES Are to synthesize primers, we need to know the sequence flanking the DNA segment of interest Only applies to short DNA fragments, mostly less than 5 kb 6. STEPS OF PCR 1. Denaturation- The melting of DNA into separate strands 2. Annealing- Primers bind to the complementary sequences on the lone strands of DNA 3. Extension- Continuation of annealing, creates copies Repeat 7. Denaturation Heating up DNA Splitting into two strands 94 degrees Celsius 8. Annealing Cooling Down Allowing to be bonded (primers) 54 degrees Celsius 9. Extension Taq Enzyme creating copy of original DNA Two new double stranded DNA formed 72 degrees Celsius 10. PCR PROGRAM 11. 1. DNA Template: DNA segment to be amplified 2. Primers: a short segment of DNA needed as starter for synthesize DNA Base composition: G+C content between 40% to 60%. Length: 18-25 nucleotides long. Members of a primer pair should not differ in length by >3 bp. Complementarity: The 3 terminal sequence of one primer should not be complementary to any site on the other primer. Melting temperature (Tm): The calculated Tm values of a primer pair should not differ by >5C. 3 termini: If possible, the 3 base of each primer should be G or C. REACTION MIXTURE FOR PCR 12. Wallace rule: This equation can be used to calculate the Tm of duplexes 15-20 nucleotides in length in solvents of high ionic strength (e.g. 1M NaCl). Tm (in C) = 2 (A+T) +4 (G+C) Calculating the melting temperature 3. Buffer every enzyme requires some conditions in means of pH, ionic strength, present cofactors etc. So, by adding the buffer to PCR reaction you get the optimal pH and Mg2+ is required as cofactor as by most NTP-binding proteins REACTION MIXTURE FOR PCR 13. Magnesium Chloride is required for Taq Polymerase to function effectively. When the enzyme polymerase binds to the DNA strand, it requires magnesium ions with hydroxide groups to remove a hydrogen proton from the deoxyribose of the nucleotide, in order to add the next nucleotide. 4. MgCl2 REACTION MIXTURE FOR PCR 5. Taq Polymerase: This enzyme will be needed to synthesize DNA copies. 6. dNTPs: Building blocks for new DNA strand. 7. Distilled Water: 14. TYPES OF PCR Allele-specific PCR Assembly PCR PCA (Polymerase Cycling) Assembly PCR ( Asymetric PCR) Helicase - Dependent amplification Hot-start PCR Inter sequence specific PCR ISSR Inverse PCR Ligation-mediated PCR Methylation - specific PCR (MSP) Mini Primer PCR Multiplex PCR Multiplex PCR Multiplex Ligation- dependent Probe Amplification MLPA Nasted PCR Overlap Extension PCR Quantitative PCR (Q-PCR) Reverse Transcription PCR Solid Phase PCR Thermal asymmetric interlaced PCR (TAIL PCR) PAN-AC Touch Down PCR Universal Fast Walking RFLP-PCR(PBR) SSCP-PCR SOEING RAPD-PCR Arbitraily primed - PCR ARMS- PCR Real-Time-PCR BOOSTER-PCR DAF-PCR SCAR-PCR AFLP a ALP,ST 15. Reverse Transcriptase-PCR RT-PCR, one of the most sensitive methods for the detection and analysis of rare mRNA transcripts or other RNA present in low abundance. RNA cannot serve as a template for PCR, so it must be first transcribed into cDNA with reverse transcriptase from Moloney murine leukemia virus or Avian myeloblastosis virus, and the cDNA copy is then amplified. The technique is usually initiated by mixing short (12- 18 base) polymers of thymidine (oligo dT) with messenger RNA such that they anneal to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the oligo dT as a primer to synthesize so-called first-strand cDNA. Roche Molecular Biochemicals: PCR Application Manual. RT-PCR 16. Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment. The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. Nested PCR is a very specific PCR amplification. 17. Hot Start PCR Hot Start PCR significantly improve specificity, sensitivity and yield of 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., 95C) 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. 18. Real-Time PCR 19. APPLICATIONS OF PCR Molecular Identification Sequencing Genetic Engineering Molecular Archaeology Bioinformatics Site-directed mutagenesis Molecular Epidemiology Genomic Cloning Gene Expression Studies Molecular Ecology Human Genome Project DNA fingerprinting Classification of organisms Genotyping Pre-natal diagnosis Mutation screening Drug discovery Genetic matching Detection of pathogens 20. WHY ELECTROPHORESIS? To separate DNA fragments from each other To determine the sizes of DNA fragments To determine the presence or amount of DNA To analyze restriction digestion products 21. PRINCIPLE separates molecules from each other on the basis of size and/or charge and/or shape basis of separation depends on how the sample and gel are prepared 22. MATERIAL REQUIRED FOR AGAROSE GEL ELECTROPHORESIS Electrophoresis chamber Agarose gel Gel casting tray Buffer Staining agent (dye) A comb DNA ladder Sample to be separate 23. Method For Electrophoresis Add running buffer, load samples and marker Run gel at constant voltage until band separation occurs Pour into casting tray with comb and allow to solidify View DNA on UV light box and show results Prepare agarose gel Melt, cool and add Ethidium Bromide. Mix thoroughly. 24. WHAT PERCENTAGE GEL? Agarose Concentration in Gel (% [w/v]) Range of Separation of Linear DNA Molecules (kb) 0.3 5-60 0.6 1-20 0.7 0.8-10 0.9 0.5-7 1.2 0.4-6 1.5 0-2-3 2.0 0.1-2 If you add 2gr Agarose into 100mL water, it will give you 2% Agarose Gel) If you add 4gr Agarose into 200mL water you will also have 2% Agarose Gel . 25. Standar d DNA 2 ul PCR product 5 ul Loading dye 2 ul Loading dye 2 ul ddH2O 8 ddH2O 5