POLYMERASE CHAIN REACTION

A new technique that made it possible to synthesize large quantities of a DNA fragment without cloning it was called Polymerase Chain Reaction.

In PCR, oligonucleotide sequences identical to those flanking the targeted sequence are first synthesized. These synthetic oligonucleotides are usually about 20 nucleotides long and serve as primers for DNA synthesis. Pieces ranging in size from less than 100 bp to several 1000 bp in length can be amplified, and only 10–100 pmol primer is required. The concentration of target DNA can be as low as 10–15 uL.

The reaction mix for PCR contains :

???(a) the target DNA

???(b) a very large excess of the desired primers

??(c) a thermostable DNA polymerase and

??(d) four deoxyribonucleoside triphosphates.

The PCR cycle takes place in three steps as follows-

Step 1: The target DNA containing the sequence to be amplified is heat denatured to separate its complementary strands. Normally the target DNA is between 100 and 5000 bp in length.

?Step 2: The temperature is lowered so that the primers can anneal to the DNA on both sides of the target sequence. Because the primers are present in excess, the targeted DNA strands normally anneal to the primers rather than to each other.

Step 3: DNA polymerase extends the primers and synthesizes copies of the target DNA sequence using the deoxyribonucleoside triphosphates.

At the end of one cycle, the targeted sequences on both strands are copied. When the three-step cycle is repeated, the four strands from the first cycle are copied to produce eight fragments. The third cycle yields 16 products. Theoretically, 20 cycles will produce about one million copies of the target DNA sequence, and 30 cycles yield around one billion copies.

Advances in PCR technology:

PCR technology is improving continually and undergoing many changes as follows:

?a)????? Nowadays, RNA can be efficiently used in PCR procedures. The Tth DNA polymerase, a recombinant Thermus thermophilus DNA polymerase, will transcribe RNA to DNA and then amplify the DNA. Cellular RNAs and RNA viruses may be studied even when the RNA is present in very small amounts.

b)????? Also, PCR can quantitate DNA products without the use of isotopes. This allows one to find the initial amount of target DNA in less than an hour using automated equipment. Quantitative PCR is quite valuable in virology and gene expression studies.

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c)???? As mentioned earlier, the target DNA to be amplified is normally less than about 5000 bp in length. A long PCR technique has been developed that will amplify sequences up to 42 kilo bases long.

d)????? Multiplex PCR is another modification of PCR in which two or more target sequences can be demonstrated simultaneously in a single specimen at the same time. This method uses two or more primer sets designed for amplification of different targets. Multiplex is now increasingly evaluated for simultaneous demonstration of two or more pathogens in a clinical specimen.

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e)???? Real-time PCR is the most recent development. It is so named, because the PCR amplicons can be detected in real time. In fact, “real time” refers to the detection of amplicons after each PCR cycle. Several commercial instruments are available that combine PCR amplification of target DNA with detection of amplicons in the same closed vessel. Probe detection formats involve detecting fluorophores.

Applications of PCR-

The PCR and other molecular techniques have already proven exceptionally valuable in many areas of molecular biology, medicine, and biotechnology. These methods are useful to:

■ Amplify very small quantities of a specific DNA and provide sufficient material for accurately sequencing the fragment or cloning it by standard techniques.

■ Detect previously unrecognized or uncultivable micro-organisms.

■ Detect the genes responsible for drug resistance. They supplement conventional antimicrobial susceptibility testing for the detection of methicillin resistance in staphylococci, rifampicin resistance in M. tuberculosis, etc.

■ Predict and monitor response of individuals chronically infected with hepatitis B virus (HBV), hepatitis C virus (HCV), or HIV to antiviral therapy.

■ Provide useful information that may predict progression of the disease. Determination of HIV-1 viral load as a predictor of progression to AIDS is an example of such use.

■ Diagnose AIDS, Lyme disease, chlamydia, tuberculosis, hepatitis, the human papilloma virus, and other infectious agents.

■ Detect genetic diseases, such as sickle cell anemia, phenylketonuria, and muscular dystrophy.

Thanks and Regards,

Amita Rao

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