Structural Biochemistry/Polyermase Chain Reaction/How PCR is Performed
Overview
[edit | edit source]
In order to perform PCR, it requires several components and reagents. Firstly, it requires a DNA template that contains the DNA region (target) to be amplified. Secondly, it requires forward and reverse primers complementary to the 3' ends of each strand of the DNA target. These primers can be custom designed and can be used to induce mutations. Thirdly, it requires magnesium Mg2+ and a buffer to maintain stable conditions. Instead of magnesium, sometimes manganese Mn2+ can be used. Magnesium is preferred over manganese because a higher concentration of manganese increases the error rate during DNA synthesis. The buffer solution provides a suitable chemical environment for optimum activity and stability of the DNA polymerase. Fourthly, it requires deoxynucleoside triphosphates (dNTP; nucleotides containing triphosphate groups) for the polymerase to use during replication. It is the building-blocks from which the DNA polymerase synthesizes a new strand of DNA. Finally, it requires a temperature stable polymerase to polymerize DNA. PCR is commonly carried out in a reaction volume of 10-200 microliters in small reaction tube in a thermal cycler. The thermal cycler heats and cools the reaction tubes to the desire temperature for each step in PCR. It uses the Peltier effect. which permits both heating and cooling of the reaction tubes by reversing the electric current. The thin walls of the reaction tubes allow for rapid thermal equilibration.
Successfully replicating DNA of interest can be tricky with PCR. This is because great care must be taken to avoid contamination. It is important to ensure the reagants are adequately mixed and stored appropriately and appropriate temperature conditions are selected for the annealing and elongation phases due to primer and polymerase sensitivity and activity at different temperatures. Typically, PCR consists of usually 20-40 repeated temperature changes, called cycles, where each cycles usually contains three steps. It is necessary to perform many cycles because that way the end results will consist mainly of the desired DNA. The temperature used and the length of time they are applied in each cycle depend on the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature of the primers.
After the reagants have been mixed in together in PCR tubes (special, thin-wall, eppendorf tubes to allow for efficient conductivity of heat), the thermal cycler must be programed for various times, temperatures, and cycles. Each major step in amplification (denaturing, annealing, and elongation) requires different conditions appropriate for the primers and polymerase being used. The first step is the initialization step. This step heats the reaction to a temperature of 94-98 degrees Celsius. This temperature is held for one to nine minutes. This heating of the DNA causes the DNA to be denatured. The heat disrupts the hydrogen bonds that hold the nitrogenous bases in the double-stranded DNA together, yielding single-stranded DNA molecules. The second step is the annealing step that requires the temperature to be lowered to 50-65 degrees Celsius to allow for annealing of the primers to the single-stranded DNA template. The annealing temperature is typically 2-3 degrees Celsius below the melting temperature of the primers. In the annealing step, the polymerase binds to the primer-template hybrid and begins DNA formation. The last step in PCR is the elongation step. This step also occurs at a certain temperature depending on the polymerase being used. Although some polymerases function better at slightly different temperatures, Taq polymerase is commonly used in PCR, thus a temperature of 72 degrees Celsius is used with this enzyme. During this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the extending DNA strand. The duration for elongation depends on the DNA polymerase used and on the length of the DNA fragment to be amplified. At its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. After the last PCR cycle, a single step of final elongation is required to ensure that any remaining single-stranded DNA is fully extended. This step occurs at 70-74 degrees Celsius for 5-15 minutes. If necessary, lowering the temperature to 4-15 degrees Celsius for an indefinite time can allow for short-term storage of the reaction. Furthermore, some steps take longer than others, and each step must be timed appropriately. Once the appropriate conditions have been established, the number of cycles can be set and the PCR run. Through these steps, DNA is readily and quickly amplified. This is because the products of every PCR cycle is used again as a template in the next cycle. The resulting DNA can then be sequenced for accuracy and then studied through other techniques such as Southern blotting or agarose gel electrophoresis. It allows for size separation of the PCR products. The size of PCR products is determined by comparison with a DNA ladder (a molecular weight marker. This DNA ladder contains DNA fragments of known size. It is run on the gel alongside the PCR products to compare the sizes of the PCR products with the sizes on the DNA ladder.
Sample PCR Procedure
[edit | edit source]PCR
- Pipet everything below into a small tube.
40.5 цL H2O 5 цL 5x thermopol buffer 1 цL dNTP mix (10mM) 1.25 цL Forward primer 1.25 цL Reverse primer +1 цL DNA =50 цL rxn +1 цL pfu Turbo polymerase (add at end. keep on ice)
- Put into Thermocycler (continues with the denaturation, annealing, and elongation steps).