Real Time PCR For Your Instant Results

2007-07-18 16:23:30

( Education )



The Polymerase Chain Reaction (PCR) is a technique scientists use to make millions of copies of a DNA segment. This is important if you are interested in analyzing DNA, since most methods require large amounts of the molecule.

PCR is patterned after the natural process of DNA replication. This time, a small plastic tube serves as the cell; while reagents and a machine called a thermal cycler act like a cell’s enzymes.

How does PCR work? A PCR mixture will contain the following: free nucleotides to build a DNA strand, single stranded DNA fragments called primers to get the building started, a DNA polymerase that can synthesize the new strand even at high temperatures; magnesium chloride to make the polymerase work efficiently; and a buffer solution to maintain the mixture’s pH. The DNA template will be added to this mixture.

There are three steps involved in PCR: denaturation, where high temperatures unwind the double stranded DNA; annealing, where primers bind to the DNA strands at specific regions; and elongation, where DNA polymerase attaches free nucleotides, extending the primers and making a copy of a fragment of the template DNA strand. This cycle is repeated 30-40 times. At the end of PCR, the DNA fragment of interest will be amplified a million times its initial concentration.

PCR takes hours to complete, and, at the end of it, scientists might not even be sure if they have amplified a DNA sequence to a usable amount. To aid in research, scientists have developed a method to both amplify DNA and check its concentration during the amplification process. This is done through real time PCR.

Real Time PCR

The real time polymerase chain reaction, or real time PCR, is similar to ordinary PCR. The Real Time PCR protocol, however, involves the use of a PCR mixture with fluorescent dyes, which bind to the double stranded DNA; and modified primers which are bound to fluorescent probes. These fluorescent dyes absorb light at a certain wavelength, and it is the light signal from these dyes that the real time PCR machine picks up.

In real time PCR, the higher the concentration of fluorescent dyes, the greater the concentration of amplified DNA in the PCR mixture.

The Uses of Real Time PCR

Real Time PCR has many uses in the laboratory. It can allow you to know how the expression of a gene changes over time; and if gene expression in tissues changes in response to external cues, such as poisons, or a rise or fall in temperature. With this new, special tool, scientists the world over can track the activities of genes, find out how they can improve organisms, and even discover drugs that can make humans healthier.


All rights Reserved © Tradenet Services srl
Do not duplicate or redistribute in any form.