For PCR which of the following is not required:
Radiolabelled DNA probe Polymerase Chain ReactionPCR is a cell-free, test tube method for amplifying a target sequence of DNA. It is much faster and more sensitive than cell-based cloning.Materials required:Two primers, each about 20-35 bases long with sequence complementary to the sequence immediately adjacent to the DNA segment of interest ( flanking sequence) . DNA polymerase (e.g., Taq polymerase) which can sustain high temperature (> 600 C). A large number of free deoxynucleotides (dNTPs)The target DNA fragment.Procedure:There are three major steps in a PCR, which are repeated for 20 or 30 cycles.a. Heat Denaturation- The DNA sample to be amplified is first heated to separate the two strandsb.Annealing- The primers bind to the two separated strands of DNA ( one for each strand ) flanking the target sequence.c.Extension- Each strand is copied by the DNA polymerase, staing at the primer site. The two DNA strands each serve as a template for the synthesis of new DNA from the two primers.The above three steps are repeated again and again . The number of copies doubles in each cycle. Typically 20-30 cycles are run during this process. There is an exponential increase in the amount of target DNA, amplifying it by a million-fold to a billion-fold.By using a heat stable DNA polymerase( Taq polymerase ), the polymerase is not denatured and, therefore does not have to be added at each successive cycle.Advantages:The major advantage of PCR over cloning as a mechanism for amplifying a specific DNA sequence aresensitivity : DNA sequences present in only trace amounts can be amplified to become the predominant sequencespeedless technically difficult than traditional cloning methods using recombinant DNA techniques. Disadvantages:To synthesize primers, we need to know the sequence flanking the DNA segment of interest.Applies only to sho DNA fragments, typically less than 10 kb.
Taq polymerase
d-NTP
Primer
Radiolabelled DNA probe