what is the role of PCR in molecular diagnosis?
PCR helps in detection of infectious agents by virtue of specific genes. It helps in early detection of HIV (causing AIDS), even before the onset of disease. The specific primers to the targeted sequences in the DNA of a virus are used to amplify the viruses nucleic acid after it has been extracted from the blood. Then, the amplified viral DNA can be quantified.
PCR is also used to perform to diagnosis of cancers like leukemia and lymphomas. PCR in this case works by detecting translocation, gene mutation in genomic DNA of malignant cells of suspected cancer patients. PCR can amplify a specific DNA fragment with mutation by using specific primers and the result of PCR can be than detected on the gel with electrophoresis.
Ok, see here. I'll explain in the most simple language. PCRStands for Polymerase chain reaction. It is a technique used for the amplification of the desired genes. Why?? It's because the gene may be required for any reason, be it DNA fingerprinting comparision. It works on the basic principle of denaturation. It's simple. DNADenatures at tempratures of around 80 C. By denaturing, we mean the 2 strands open up or uncoil. Now, if we cool it, it recoils back, because the double strand is energetically more favourable. So now, it is simple. We 1st heat it, then just add some oligonucleotides of known sequences. these oligonucleotides will find their complementary sequences in the DNA strands and will bind to them. These oligonucleotides are taken such that they attach to the gene we need. Now we simply add DNA Polymerase obtained from the bacterium T. aquaticus. The special feature of this polymerase is that it works even at high tempratures of aound 80 C and doesn't denature or break down itself due to the heat. Then add nucleotides and the Polymerase will effectively copy both DNA Strands or replicate them. Thus from 2 strands, we get 4. again by cycle of denaturation, adding oligonucleotides, Stable DNAPolymerase and nucleotides, we can make the strands replicate continuously. Thus in a short time, we can amplify the frequency of thegene required by a billion times or more. This works on the simple principle that stable DNA Polymerase works around 80 C, and if provided nucleotides, it just keeps on replicating DNA and very soon (Since the gene is merely copied, it nearly remains same in all DNA copies) we obtain a workable amount of DNA, which means errors can be reduced as the required gene is present in much higher frequencies. Also, we simply cannot let the DNA cool because in double stranded state, the DNAPolymerase cannot make the DNA replicate andit becomes a double strand, which stops replicating. The purpose of denaturation of DNA is to keep the single strand configuration, which can be replicated effectively by the DNApolymerase if nucleotides are provided to it. Also, the DNAhas to be cooled before the replication, so that replication takes place. In short, we have to heat again and cool, then heat again and cool again,in cycles.