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Application of RAPD in Molecular BiologyBY: Lakshmi K Sugavanam | Category: Biotech-Research | Submitted: 2011-04-09 18:45:08
Article Summary: "Why Random Amplification of Polymorphic DNA (RAPD) is an important Molecular Biology technique. It is a PCR reaction in which the amplified target DNA is unknown. A large genetic material (DNA) of unknown origin or composition can be effectively profiled using this technique..."
RAPD (pronounced "rapid") is an abbreviation for Random Amplification of Polymorphic DNA, a popular Molecular Biology tool. It is a PCR reaction in which the amplified target DNA is unknown. A large genetic material (DNA) of unknown origin or composition can be effectively profiled using this technique. The primers used in the PCR reaction are short arbitrary primers that are about 8- 10 bases long. The template DNA is a large genome so that the arbitrary primers will, in most cases, be able to find a complementary sequence. The resulting products can be run on an agarose gel and the pattern obtained will be an almost unique genetic profile. The pattern of amplified sequences could be species or strain specific and constitute an identity profile of the organism. It can also be used to compare different strains of an organism or to compare DNA from two different sources. These features of the RAPD assay make it possible to detect DNA polymorphism in the absence of specific nucleotide sequence information, thereby avoiding much labor-intensive training and work.
For this process to be carried out, no knowledge of the target DNA sequence is required. So, it is popularly used for the comparison of the DNA extracted from unknown or comparatively new biological samples. RAPD has also been used to trace the phylogeny of diverse species of flora and fauna.
RAPD is unlike PCR analysis in that it does not need knowledge of the DNA sequence of the target organism. The ten base primers may amplify a segment of DNA, depending on whether any part of the target DNA is complimentary to the primer or not. If the primers anneal at different places instead of the complimentary sequences, no DNA is amplified. Also, if the 3' ends of the primer are not facing each other, they will not amplify the target DNA. If there is any mutation on the template DNA, the primer may not bind and a new different DNA pattern will be produced.
RAPD technology was first developed for fast gene mapping in species of economic interest. It is mostly used in studying microbial diversity, population genetics and molecular phylogenetics, because of its simplicity, speed and effectiveness at detecting polymorphism. RAPD is useful for selection of variants in the cultivable isolates because bacteria may be similar in morphology but vary in their genetic make up or they may be similar genetically but differ morphologically.
-It has several advantages over other techniques. It can be used with uncharacterized genomes and can be applied to cases in which only small quantities of DNA are available.
-Absolutely no knowledge of the target genome is required. It can be used on any DNA sample.
-RAPD is an inexpensive yet powerful tool for typing of bacterial species.
-Even if the same primer is used with different samples, they will produce different results and different bands patterns that may allow for recognition of the various strains.
-Can be used to study genetic polymorphism between closely related species.
-Can be used to select variants of microbial isolates.
-The primer sequence has to be right to produce the right results.
-Since the primer targeting is random, it is absolutely essential to have a large genome template.
-Minute quantities of available DNA or degraded DNA samples cannot be subjected to RAPD (unlike PCR). It is very likely that the primer will not be able to find complementary sequences on the target.
-It has a low power of resolution unlike other methods of DNA analysis.
-The quality of the DNA used in the reaction will affect the outcome.
-The concentration of PCR reagents, their purity, and the conditions in which the reaction is carried out will all affect the results.
-RAPD requires knowledgeable and careful operation for good results and for these results to be reproducible.
-If there is a mismatch between the primer and the template DNA, there will be reduced product or even no PCR product. Hence, reading the results can be a little tricky sometimes.
-If the 3' ends of the primer are not facing each other there will be little or no product.
-Mutation of target DNA can give wrong profile.
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