![]() Extension/elongation step: The temperature at this step depends on the DNA polymerase used Taq polymerase has its optimum activity temperature at 75-80☌, and commonly a temperature of 72☌ is used with this enzyme.Annealing step: The reaction temperature is lowered to 50-65☌ for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template.It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98☌. ![]() The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle. The following are the steps of PCR: Figure: The Steps of PCR: This illustrates a PCR reaction to demonstrate how amplification leads to the exponential growth of a short product flanked by the primers. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers. The temperatures used, and the length of time they are applied in each cycle, depend on a variety of parameters. Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of two to three discrete temperature steps, usually three. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.Divalent cations, magnesium or manganese ions generally Mg2 .Deoxynucleoside triphosphates (dNTPs nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.Taq polymerase or another DNA polymerase with a temperature optimum at around 70 ☌.Two primers that are complementary to the 3′ (three prime) ends of each of the sense and anti-sense strand of the DNA target.DNA template that contains the DNA region (target) to be amplified.A basic PCR set up requires the following components and reagents: The reaction produces a limited amount of final amplified product that is governed by the available reagents in the reaction, and the feedback-inhibition of the reaction products. ![]() Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size. PCR is used to amplify a specific region of a DNA strand (the DNA target). PCR can be extensively modified to perform a wide array of genetic manipulations. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. Primers (short DNA fragments) containing sequences complementary to the target region, along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. ![]() The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |