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Friday, March 4, 2011

Polymerase chain reaction PCR

What is Polymerase chain reaction (PCR)?
Polymerase chain reaction (PCR) is a technique in biotechnology and molecular biology. It has various applications in medicine and diagnosis of a diseases, it is also used for DNA cloning and sequencing and genetic functional analysis, genetic manipulations, genetic finger printing and diagnosis of genetic disease, paternity testing, and to identify the presence of genetic material of an infection or the presence of a microorganism in a given test sample by finding out the presence of its genetic material that is DNA.

The beauty of this technique is that, it can find out even a single copy of the DNA or even in fraction of the DNA of an infectious organism in the test samples, and then this technique amplifies the presence of fraction of a copy of genetic material of the microorganism by allowing it to multiply and polymerize in to several numbers to the tune of several thousand copies which are then marked with a visible marker so as to make the result of the test visible.

Single DNA strand in the test sample which is used as a template for the polymerization reaction, is incubated with a DNA polymerase enzyme, and a sequence of heating and cooling cycles are applied, due to which DNA strands are opened in to single strands and the open strands are further acted up on by DNA polymerase enzyme to produce more number of complementary copies of DNA strands, the reaction make use of DNA oligonucleotides which contains building blocks of the DNA that is nucleotides which are also called as DNA primers.

The DNA polymerase enzyme usually employed in polymerase chain reaction (PCR) is a heat stable polymerase enzyme called as Taq polymerase which was derived from T. aquaticus a bacteria which grows in hot water springs , Taq polymerase is capable of withstanding the high temperature which is employed while melting of the DNA present in the test sample a while conducting a polymerase chain reaction (PCR).
The newly formed complementary DNA strand further serve a new template and also take part in further reaction to produce more number of copies of DNA , thus amplifying the reaction several folds exponentially.
Since the Polymerase chain reaction (PCR) make use of basic unit of any cell that is DNA to identify the infection, therefore it is considered more reliable than the tests like ELISA which make use of antibodies produced against the disease, as presence of antibodies in once blood solely does not confirm the presence of disease, as antibodies to particular diseases may be produced because of certain other diseases or vaccinations.

Polymerase chain reaction (PCR) is used as one of the final confirmatory tests in diagnosis of HIV infection, while ELISA may give falls negative result in latency period in HIV infected individual where the sufficient amount of antibodies against HIV are not formed in ones body immediately after contracting with HIV virus, where as polymerase chain reaction (PCR) can even diagnose the presence of HIV virus in blood of the infected individual immediately.

What is reverse Transcription PCR (RT PCR): 
In PCR a genetic material is formed from DNA, while in case of RT PCR DNA is formed from mRNA formation of complementary strand of DNA is carried out by mRNA as in conventional its done from DNA itself. Complementary DNA strand makes its double strand, this reaction work in a chain.

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