Polymarase Chain Reaction PDF

Polymarase Chain Reaction Biochemical and Genetic Techniques Lab 8 Three stage Polymarase Chain Reaction Polymerase Chain Reaction (PCR): serves a crucial role in molecular biology and genetics research. The primary purpose of PCR is to amplify a specific regi...

Polymarase Chain Reaction Biochemical and Genetic Techniques Lab 8 Three stage Polymarase Chain Reaction Polymerase Chain Reaction (PCR): serves a crucial role in molecular biology and genetics research. The primary purpose of PCR is to amplify a specific region of DNA, producing millions to billions of copies of that particular DNA segment. This has various applications in scientific research, diagnostics, forensics, and more. The PCR reaction requires the following components TEMPLATE - the purified, double-stranded piece of DNA we want to copy PRIMERS - short synthetic DNA molecules that target a specific DNA sequence for amplification TAQ DNA POLYMERASE - thermostable enzyme used to copy DNA FREE NUCLEOTIDES - the building blocks of DNA THERMAL CYCLER (a.k.a. PCR machine) - a specialized machine that rapidly heats and cools the samples Buffer: The reaction buffer provides the optimal conditions for the PCR reaction, including the appropriate pH and salt concentration. MgCl2: Magnesium ions are cofactors for DNA polymerase and are essential for its enzymatic activity. Protocol: A. Prepare Reaction Mix: In a 0.5-ml tube or 0.2-ml PCR tube, add the following on ice: 1. 1 μl of dsDNA template (~0.1 μg) 2. 2 μl of 10X buffer 3. 1 μl of 4 mM dNTP mix 4. 1 μl of 10 μM forward primer (to a final concentration of 0.5 μM) 5. 1 μl of 10 μM reverse primer (to a final concentration of 0.5 μM) 6. 1 μl of 1 unit/μl Taq polymerase 7. 13 μl of DNA and DNase-free water B. Mixing and Centrifugation: Tap the tube gently to mix the contents. Briefly spin the tube in a microcentrifuge to ensure all contents are at the bottom.  C. Preparation for Thermocycler: Keep the prepared reaction mix on ice until ready to load into the thermocycler. D. Loading Thermocycler: If the thermocycler has a heated lid, proceed without additional steps. If the thermocycler does not have a heated lid, layer a thin film of mineral oil over the reaction mix to prevent evaporation. E. PCR Amplification: Set up the appropriate PCR program on the thermocycler based on the requirements of your specific PCR reaction. F. Post-PCR Handling: After the PCR reaction is complete, store the amplified products appropriately or proceed with downstream applications. The basic steps of a PCR procedure Denaturation: Objective: Separate the DNA strands. Temperature: Typically 94-98°C. Time: 20-30 seconds. Description: Heat the reaction mixture to near boiling temperatures to break the hydrogen bonds between complementary DNA strands, resulting in the denaturation of double-stranded DNA into two single strands. Annealing: Objective: Allow primers to bind to the complementary sequences on the DNA template. Temperature: Typically 50-65°C. Time: 20-40 seconds. Description: Cool the reaction mixture to a temperature that allows specific primers to anneal (bind) to their complementary sequences on the single-stranded DNA template. This step is crucial for the specificity of the PCR reaction. The basic steps of a PCR procedure Extension (Elongation): Objective: DNA synthesis by the DNA polymerase enzyme. Temperature: Typically 68-72°C. Time: Time depends on the polymerase used and the length of the DNA fragment. Description: The DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand. The temperature is usually higher to facilitate the activity of the DNA polymerase. Final Extension: Temperature: Typically 68-72°C. Time: 5-10 minutes. Description: This step allows any remaining single-stranded DNA to be fully extended and ensures that all the target DNA is copied. It also helps in the completion of any partial DNA strands. The basic steps of a PCR procedure PCR Cycle Repetition: Number of Cycles: Typically 20-40 cycles. Description: The denaturation, annealing, and extension steps are repeated for the specified number of cycles. Each cycle doubles the amount of DNA in the reaction, leading to exponential amplification of the target DNA. Hold at 4-10°C: Temperature: 4-10°C. Description: The reaction is held at a low temperature to stabilize the amplified DNA. After completing the PCR, the amplified DNA can be analyzed through various techniques such as gel electrophoresis or sequencing.  The equation for calculating the number of PCR cycles can be expressed as follows: Number of Copies=Initial Copies×(2)Number of CyclesNumber of Copies=Initial Copies×(2)Number of Cycles Here's an example to illustrate how to use this equation: Let's say you start with one copy of a DNA fragment, and each PCR cycle doubles the number of copies. After 20 cycles, you want to calculate how many copies you would have: Number of Copies=1×(2)20 Number of Copies=1×(2)20 Number of Copies=1×220 Number of Copies=1×220 Number of Copies=1×1,048,576 Number of Copies=1×1,048,576 So, after 20 PCR cycles, starting with just one copy, you would have approximately 1,048,576 copies of the DNA fragment. Thank you