Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction

The polymerase chain reaction (PCR), invented by Kary Mullis in the early 1980s, exploded onto the biotechnology landscape. It changed the way genes are cloned, nucleic acids sequenced, diseases diagnosed, and crimes solved. Why is PCR so versatile and important? Quite simply, it enables the rapid synthesis of billions of copies of a specific DNA fragment from a complex mixture of DNA. Researchers can thus obtain large quantities of specific pieces of DNA for experimental and diagnostic purposes.

How PCR works?

Suppose that one wishes to make large quantities of a particular DNA sequence, a process known as gene or DNA amplification. The first step is to synthesize oligonucleotides (Greek oligo, few or scant)-single stranded DNA fragments with sequences complementary to those flanking the targeted sequence. Oligonucleotides are made with a DNA synthesizer and are generally between 15 and 30 nucleotides long. Oligonucleotides serve as DNA primers, providing the 3' -OH needed for DNA synthesis during PCR. The primers are added to the reaction mixture, along with the template DNA (often copies of an entire genome), a thermostable DNA polymerase, and each of the four deoxyribonucleoside triphosphates (dNTPs).
PCR Components
PCR requires a series of repeated reactions, called cycles. Each cycle has three steps that are precisely executed in a machine called a thermocycler. In the first step, the DNA containing the sequence to be amplified is denatured by raising the temperature to about 95°C. Next the temperature is lowered to about 50°C so that the primers can hydrogen bond (anneal) to the DNA on both sides of the target sequence. 

Finally, the temperature is raised, usually to 68 to 72°C, so that DNA polymerase can extend the primers and synthesize copies of the target DNA sequence using dNTPs. Only polymerases that function at the high temperatures can be used. The most commonly used thermostable enzyme is Taq polymerase from the thermophilic bacterium Thermus aquaticus.

At the end of one PCR cycle, the targeted sequences on both strands have been copied. When the three-step cycle is repeated, the two strands from the first cycle are copied to produce four fragments. These are amplified in the third cycle to yield eight double-stranded products. Thus, each cycle increases the number of target DNA molecules exponentially. Depending on the initial concentration of the template DNA and other parameters such as the G + C content of the DNA to be amplified, it is theoretically possible to produce about 1 million copies of targeted DNA sequence after 20 cycles and over 1 billion after 30 cycles. 
Polymerase Chain Reaction (PCR)
PCR is most frequently used in two ways. If large quantities of a specific piece of DNA are needed, the reaction products are collected and purified at the end of a designated number of cycles. This is sometimes called end-point PCR, and the final number of DNA fragments amplified is not quantitative. This means that the amount of final product does not always reflect the amount of template DNA present. 

In contrast, real-time PCR is quantitative; in fact, it is referred to as qPCR. That is, the amount of DNA or RNA template (which is converted to DNA with reverse transcriptase prior to starting PCR) present in a given sample can be determined. This is accomplished by adding a fluorescently labeled probe to the reaction mixture and measuring its signal during the initial cycles.
PCR Machine
This is when the rate of DNA amplification is logarithmic. However, as the PCR cycles continue, substrates are consumed and polymerase efficiency declines. So although the amount of product increases, its rate of synthesis is no longer exponential (this is why end-point collection of PCR products is not quantitative). 

Specially designed thermocyclers record the amount of PCR product generated as it occurs, thus the term real-time PCR. Gene expression studies often rely on real-time PCR because mRNA transcripts can be copied by reverse transcriptase to eDNA, which is then quantified. Therefore the procedure monitors transcription of the gene targeted by the primers.

Application of polymerase chain reaction:

  1. PCR is an essential tool in many areas of molecular biology, medicine, and biotechnology. when PCR is used to obtain DNA for cloning, a number of steps in traditional cloning procedures are no longer required.
  2. PCR is also used to generate DNA for nucleotide sequencing because the primers used in PCR target specific DNA, PCR can isolate particular fragments of DNA (e.g., genes) from solutions that contain many different genomes, such as soil, water, and blood.
  3. PCR is used to amplify specific genes from the environment without first culturing members of the microbial community. 
  4. It has also become an essential part of certain diagnostic tests, including those for AIDS, Lyme disease, chlamydia, tuberculosis, hepatitis, human papillomavirus, and other infectious agents and diseases.
  5. PCR is also employed in forensic science, where it is used in criminal cases as part of DNA fingerprinting technology.
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