Friday, May 18, 2012

Polymerase Chain Reaction (PCR) a diagnostic tool.

What is PCR?

Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast and inexpensive technique used to amplify, or make many copies of, small segments of DNA. This is necessary because methods used for analyzing DNA (determining the DNA base pair sequence) require more DNA than may be in a typical sample. A particularly useful feature of PCR is that it allows the amplification process to be limited to specifically targeted segments of the DNA mixture--such as the Y chromosome markers used in genealogical testing.
When your vials of cheek cells arrive at the lab for testing, they are first mixed with a detergent which causes the cells to burst open and release their DNA along with other cell contents. The mixture is then washed with a phosphate containing buffer (mild salt) solution to dilute cellular debris. With minimal preparation, the sample is ready and the DNA on the targeted area of a chromosome can be amplified.

How does PCR work?

PCR is a process based on the ability of a DNA polymerase enzyme that can synthesize a complementary strand to a targeted segment of DNA in a test tube mixture of the four DNA base. In addition, the mixture must also contain two DNA fragments, each about 20 bases long, called primers, that have sequences complementary to areas adjacent to each side of the target sequence. (To do PCR, you need to know the DNA sequence around the region you want to amplify.) These primers can be constructed in the lab, or purchased from commercial suppliers. If chosen well, the 20-25 base pair sequence will be unique in the entire human genome so will match only the place specifically chosen thus limiting and defining the area to be copied.
The mixture is first heated to denature (separate) the sides of the double- stranded DNA and then cooled to allow (1) the primers to find and bind to their complementary sequences on the separated strands and (2) the polymerase to extend the primers into new complementary strands. Repeated heating and cooling cycles multiply the target DNA exponentially, since each new double strand separates to become two templates for further synthesis. In about 1 hour, 20 PCR cycles can amplify the target by a millionfold. In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created.

From: The National Human Genome Research Institute Office of Science Education and Outreach

The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis. To avoid the destruction of needed enzymes in the mixtures by the high temperatures needed to denature the DNA, enzymes from bacteria that thrive in hot springs are used for the process.

Why is PCR useful?

Once amplified, PCR products can be used in many different laboratory procedures; for example, most mapping techniques in the Human Genome Project rely on PCR.
PCR is also valuable in a number of newly emerging laboratory and clinical techniques, including DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders and preparing samples for genealogical DNA testing.


No comments:

Post a Comment