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Random Amplification of Polymorphic DNA
Introduction
Genetic markers can be defined as a sequence of DNA with a known physical location on a chromosome. Genetic markers and genes that are close to each other tend to get inherited together. Genetic markers vary between individuals to the extent they can be used to help find a nearby gene causing a certain disease or trait within a family.
Random amplified polymorphic DNA is one of such genetic markers which used the polymerase chain reaction method for the amplification of random DNA segments on a chromosome.
Random Amplified Polymorphic DNA
This technique uses a short primer that attaches itself to the complementary sequence on genomic DNA and then amplifies the segment of DNA by polymerase chain reaction. This technique produces small DNA fragments which can be observed using agarose gel electrophoresis by using ethidium bromide. The primers used to act as both forward as well as reverse primers and bind themselves randomly.
Principle
Random amplified polymorphic DNA is based on the principle of the enzymatic process for the amplification of the specific DNA fragment from the DNA of interest. The whole process is known as polymerase chain reaction.
Steps Involved in RAPD
In the first step a template DNA is added to the polymerase chain reaction tubes are kept in the racks.
A reaction mixture is prepared by adding PCR buffer, sterilized water, and dNTPs. Magnesium chloride, Taq polymerase enzyme, RAPD primer, and template DNA in different concentrations in an Eppendorf tube.
If the reaction involves more than one primer, then separate reaction mixtures should be prepared. The Eppendorf tube should be kept on ice while preparing the PCR reaction mixture to avoid any denaturation of the enzyme.
The reaction mixture present in the tube is mixed with template DNA and mixed well by patting the tubes with the fingers.
After mixing it is subjected to centrifugation to bring the contents of the tube to the bottom. Here vortex mixer is not used as it produces heat which can denature the enzyme.
PCR cycle is performed by selecting a suitable number of cycles in a thermocycler.
After the completion of the PCR cycle the amplified product is determined by running it through the agarose gel containing ethidium bromide.
The PCR mixture is mixed with gel-loading dye. The commonly used gel loading dye used in agarose gel electrophoresis is bromophenol blue. Gel-loading dye imparts blue color, as well as the glycerol present in it gives weight to the mixture so that it cannot spill out of the tank.
A marker is also loaded along with the PCR mix in the well to determine the completion of the electrophoresis.
The gel is connected to the power supply and allowed to run till bromophenol blue reaches the other end. After this power supply is switched off and the gel is removed.
The gel with amplified fragments is observed under a transilluminator.
Advantages of RAPD
One of the most important advantages of this technique is that is very quick and easy to assay.
Only a very low quantity of the DNA is required as it can be amplified using a polymerase chain reaction.
No need for sequence data for the construction of the primers is required as random primers are involved in the amplification of template DNA.
Since RAPDs are distributed throughout the DNA therefore, they have very high genomic abundance.
Large number of fragments are produced in a single cycle.
Disadvantages of RAPD
The DNA to be analyzed should be of very good quality and also should be of high molecular weight and also should be purified and strict precautions should be taken to avoid any cross contamination because short primers are used that are capable of amplifying DNA in a wide variety of organisms.
This method requires a highly sophisticated experimental procedure because of its low reproducibility also enzymes used are highly sensitive to reaction conditions.
This method does not depend on the position of the fragment, so the results cannot be interpreted in terms of locus also similar-sized fragments may not be homologous.
Applications of RAPD
This technique can be used to study certain genetically linked or closely related species.
This method has been used in gene mapping studies to cover the lags which is not possible with any other molecular marker.
Some of the variants of this technique have been used to increase its sensitivity. For example, DNA amplification fingerprinting uses very short primers to produce a large number of fragments. One more variant is arbitrarily primed polymerase chain reactions which use large arbitrary primers for the amplification.
It can also be used to study population and evolution genetics.
It is used to determine the pesticide resistance in some organisms.
Conclusion
The uniqueness of RAPD lies in the fact that it does not require the knowledge of the genome sequence to be amplified and random sequences are amplified by polymerase chain reaction. Other variants of RAPD have also been used for phylogenetic studies.
But, since random sequences are amplified correct interpretation of the results becomes difficult, therefore more sensitive techniques are used to overcome this limitation.
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