The reaction temperature is rapidly lowered to 54-60☌ for 20-40 seconds. During this, the double stranded DNA is denatured to single strands due to breakage in weak hydrogen bonds. This step involves heating the reaction mixture to 94☌ for 15-30 seconds. Oligonucleotide primers : Short pieces of single stranded DNA (often 20-30 base pairs) which are complementary to the 3’ ends of the sense and anti-sense strands of the target sequence.ĭeoxynucleotide triphosphates : Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA synthesis.īuffer system : Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation also important for polymerase activity, stability and fidelity.Īll the PCR components are mixed together and are taken through series of 3 major cyclic reactions conducted in an automated, self-contained thermocycler machine. The PCR reaction requires the following components:ĭNA Template : The double stranded DNA (dsDNA) of interest, separated from the sample.ĭNA Polymerase : Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98°), and can function at a temperature optimum of about 70☌. PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments. Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. It is an in vitro method of amplifying a desired DNA sequence of any origin hundreds of million times in hours. Polymerase chain reaction (PCR) is a very versatile gene amplification method that has brought a tremendous progress in molecular biology and genetics.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |