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Laboratory work Nr.2 PCR Polymerase Chain Reaction Polymerase chain reaction (PCR) PCR discovery by Kary Mullis in 1983 allows the scientists to mimic the cell’s own natural DNA replication process in a test tube. For PCR discovery Mullis received the Nobel prize in Chemistry in 1993. PCR allows the amplification of specific DNA sequences in a large quantities from incredibly small amounts of material. This method is so original and significant, that virtually divides biology into the two epochs of before PCR and after PCR. PCR become a central laboratory method in molecular biology. DNA replication in vivo The majors steps in DNA replication process in cells : 1. Helicase unwinds the DNA helix 2. RNA primase synthesize complementary RNA primers 3. DNA polymerase attach to the primer ends 4. Synthesis of complimentary DNA fragment. DNA replication in vitro 1. dsDNA denaturation at 96⁰C 2. Synthetic DNA primers bind to DNA strand 3. DNA polymerization reaction DNA pol DNA pol DNA polymerase is a protein. DNA pol Proteins tend to denature (unfold and lose activity) at high temperatures. Therefore, in PCR we use thermostable DNA polymerases. Thermostable DNA polymerases • Isolated from the hot spring organisms. • Taq DNA polymerase was originally isolated from thermophilic bacterium Thermus aquaticus that lives in hot springs were temperatures exceed +70⁰C (found in Yellowstone national park). Halflife of Taq DNA polymerase at +95⁰C is 1.6 hours. • Pfu DNA polymerase was isolated from hiperthermophilic arhaebacterium Pyrococcus furiosus (Volcanic island in Italy) that lives at +70- +103⁰C temperatures. • Taq polymerase is faster and cheaper than Pfu polymerase. • Pfu polymerase have the lowest error rate. In vivo vs In vitro DNA replication The majors steps in DNA replication process in vivo The majors steps in DNA replication process in vitro Helicase unwinds the DNA helix DNA unwinding occurs at high temperature RNA primase synthesize complementary RNA primers Synthetic DNA primers DNA polymerase attach to the primer DNA polymerase attach to the primer ends ends Synthesis of complimentary DNA Synthesis of complimentary DNA fragment. fragment. PCR DNA in the PCR reaction would grow exponentially. 25 to 35 cycles is the standard for a PCR reaction. This results in from approximately 34 million to 34 billion copies of desired sequence, respectively. PCR primers • Synthetic single strand DNA fragments (16-25nt) that are complimentary to template DNA. • Each primer has melting temperature (Tm). Tm relies on length and composition of the primers. • Praimers annealing temperature should be 5⁰C below the lowest primer Tm. • Tm of the primers could be calculated by using formula • Tm = 2 X (A+T) + 4 X (G+C) PCR applications PCR is widely used in medical and biological research for a variety of applications • DNA amplification for DNA cloning and sequencing • For scientific and analytical objectives – genome analysis, artificial DNA construct analysis • Clinical diagnostics – detection of patogen presence in sample • Forensic medicine – to reveal the person’s identity, paternity tests • Food product analysis (example, if the meat product contains horse or cat meat) PCR laboratory work Target substrates Diabetes caused by mutations in the HNF1A (encoding hepatocyte nuclear factor-1 alpha) and GCK4 (encoding glucokinase 4) genes is one of the most common types of maturity onset diabetes of the young (MODY). HNF1α is a transcription factor that is important for the normal development of beta cells. Mutations in the HNF1A gene cause diabetes by lowering the amount of insulin that is produced by the pancreas. Mutations in HNF1A accounts for 70% of MODY cases. GCK4 is a gene that codes for an enzyme, known as glucokinase, which helps the body produce insulin in response to increases in blood sugar. Mutations in one of the two copies of the GCK4 gene result in blood sugars that are mildly elevated above normal levels. This is one of the most common forms of MODY, it is estimated to affect about 1 in every 1000 people. PCR procedure 1. Setting up the reaction mix PCR 1 tube GCK4 gene fragment amplification PCR 2 tube HNF1A gene fragment amplification PCR 3 tube Negative controlno template control (NTC) Polymerase chain reaction Procedure 1. Set up the reaction mixture: Buffer Water Primer Forward Mg2+ ions 5’- ATTGCCA-3’ ATTGCCA Primer Reverse dNTP mix 5’- TATCCGA-3’ Template DNA dATP+ dCTP + dGTP + dTTP DNA polymerase Setting up the reaction mix 1. Place 0.2ml PCR tubes on ice. 2. Set up 25 µl PCR reaction (keep all your reagents on ice). Reagents PCR 1 volume, µl PCR 2 volume, µl PCR 3, NTC volume, µl 1x 2.5mM 200 µM Sterile dH2O 10x Hot Fire DNA buffer 2.5 2.5 17.4 2.5 MgCl2 (25mM) dNTP mix (10mM) 2.5 0.5 2.5 0.5 2.5 0.5 Primer Fw (100 pmol/µl) 0.8 0.8 0.8 Primer Rev (100 pmol/µl) 0.8 0.8 0.8 Template DNA Hot FirePol DNA Polymerase (5U/µl) Total volume of reaction Final concentration - 50 ng 2.5U 0.5 0.5 0.5 25 µl 25 µl 25 µl •In PCR1 tube the primers 90 and 91 should be used for amplification of the GCK4 gene fragment. •In PCR2 tube the primers S19L Fw and S19L Rev should be used for amplification of the HNF1A gene fragment. •In PCR3 tube you can choose one primer set or another. PCR reaction 3. Set up the PCR program Initial Denaturation for 10 minutes at 95°C: In this initiation step the hydrogen bonds are broken between the nucleotide base pairs and DNA strands separate from each other. Denature 30 seconds at 95°C: Continued denaturation of DNA double helix. PCR reaction 3. Set up the PCR program Anneal primers for 30 seconds at 60°C: The forward and reverse primers anneal to each of the single stranded DNA template strands. The DNA polymerase bind to the primer DNA sequence. Extend DNA for 30 seconds at 72°C: The Taq polymerase has an optimal temperature around 70-75°C so this step enables the DNA polymerase to synthesize and elongate the new target DNA strand accurately and rapidly. Repeat steps 2-4 35 times. PCR reaction 3. Set up the PCR program Final Extension for 7 minutes at 72°C: A final extension to fillin any protruding ends of the newly synthesized strands. 4. Place the PCR tubes into PCR thermocycler and run the program. Validating the PCR reaction Once your PCR reaction has run (2h), you will determine success or failure. You will take some of the final PCR reaction and run it out on an agarose gel with an appropriate molecular weight marker to make sure that the reaction was successful and the amplified product is the expected size relative to the maker. 5. Add 5 µl of 6x Loading dye into each PCR reaction tube and vortex. Carefully load your samples into the wells of the 1% agarose gel and run the gel. PCR result analysis by agarose gel electrophoresis 4) PCR reaction analysis in agarose gel 100bp marker Electrophoresis uses an electrical field to move the negatively charged DNA toward a positive electrode through an agarose gel matrix. The gel matrix allows shorter DNA fragments to migrate more quickly than larger ones. 1. 2. 3. 4. 5. -cont. Thus, you can accurately determine the length of a DNA segment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths). Genomic DNA concentrations Genomic DNA concentration and purity DNA yield and purity could be estimated by measurement of absorbance. DNA concentration is estimated by measuring the absorbance at 260nm (A260), adjusting the A260 measurement for turbidity (A320 measurement), multiplying by the dilution factor, and using the relationship that A260 of 1.0= 50 µg/ml pure double stranded DNA. DNA purity calculate as the ratio of the absorbance at 260nm divided by the readings at 280nm. Good quality DNA will have an A260/A280 ratio 1.7-2.0. A reading below 1.7 does not render that DNA unsuitable for any application, but lower ratios indicate more contaminants are present. Your genomic DNA extraction results Genomic DNA concentrations Tube number DNA conc, ng/µl DNA purity, A260/A280 1. 58 1.78 2. 79 1.4 3. 22 1.96 4. 379 1.68 5. 25 1.33 6. 77 1.77 7. 112 1.7 8. 12 1.85 9. 89 1.84 10. 63 1.86 31 1.5 11. 12. The ideal purity for genomic DNA is in the range 1.80 – 1.89.