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Term Paper on RAPD

Term Paper # 1. Introduction to RAPD:

Advances in molecular biology techniques have provided the basis for unraveling virtually unlimited numbers of DNA markers. The utility of DNA-based markers is generally determined by the technology that is used to reveal DNA-based polymorphism. Presently, the restriction fragment length polymorphism (RFLP) assay has been the choice for many species to measure genetic diversity and construct a genetic linkage map.

However, an RFLP assay which detects DNA polymorphism through restriction enzyme digestion, coupled with DNA hybridization, is, in general, time consuming and laborious. Over the last decade, polymerase chain reaction (PCR) technology has become a widespread research technique and has led to the development of several novel genetic assays based on selective amplification of DNA.


This popularity of PCR is primarily due to its apparent simplicity and high probability of success. Unfortunately, because of the need for DNA sequence information, PCR assays are limited in their application. The discovery that PCR with random primers can be used to amplify a set of randomly distributed loci in any genome facilitated the development of genetic markers for a variety of purposes.

The simplicity and applicability of the RAPD technique have captivated many scientist interests. Perhaps the main reason for the success of RAPD analysis is the gain of a large number of genetic markers that require small amounts of DNA without the requirement for cloning, sequencing or any other form of the molecular characterization of the genome of the species to be analyzed.

Therefore, Random Amplified Polymorphic DNA (RAPD) markers are DNA fragments obtained by PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence.

Term Paper # 2.Principle of RAPD:


RAPD technique uses short synthetic oligonucleotides (approximately 10 bases long) of random sequences as primers to amplify small amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are then separated on agarose gels and stained with ethidium bromide.

Welsh and McClelland independently developed a similar technique using primers approximately 15 nucleotides long and different amplification and electrophoretic conditions from RAPD and called it the arbitrarily primed polymerase chain reaction (AP-PCR) technique.

PCR amplification with primers shorter than 10 nucleotides [DNA amplification fingerprinting (DAF)] has also been used producing more complex DNA fingerprinting profiles. At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other.

The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNA will result in the presence or absence of bands because of changes in the priming sites. The profile of RAPD bands is similar to that of low stringency mini-satellite DNA fingerprinting patterns and is therefore also termed RAPD fingerprinting. RAPD are dominant markers.


Term Paper # 3. Applications of RAPD:

RAPD is a simple and cost effective technique because of which it has found a wide range of applications in many areas of biology.

Some of the areas where the technique is used are as follows:

1. Genetic Mapping:


Restriction fragment length polymorphisms (RFLPs) have been commonly used to map genes. This approach involves hybridization of a probe to Southern blotted genomic DNA digested with restriction endonucleases. A useful probe will detect differences in restriction fragment lengths arising from loss or gain of recognition sites or from deletions or insertions of stretches of DNA between sites. The speed and efficiency of RAPD analysis encouraged scientists to perform high-density genetic mapping in many plant species such as alfalfa, faba bean and apple in a relatively short time.

2. Developing Genetic Markers Linked to a Trait:

One of the most widely used applications of the RAPD technique is the identification of markers linked to traits of interest without the necessity for mapping the entire genome.

3. Population and Evolutionary Genetics:


The RAPD technique has received a great deal of attention from population geneticists because of its simplicity and rapidity in revealing DNA-level genetic variation, and therefore has been praised as the DNA equivalent of allozyme electrophoresis.

A major drawback of RAPD markers in population genetic studies of outbreeding organisms is that they are dominant. Thus gene frequency estimates for such loci are necessarily less accurate than those obtained with co-dominant markers such as allozymes and RFLPs.

4. Reproducibility of RAPD Markers:

RAPD reaction is far more sensitive than conventional PCR because of the length of a single and arbitrary primer used to amplify anonymous regions of a given genome. This reproducibility problem is usually the case for bands with lower intensity. Perhaps some primers do not perfectly match the priming sequence, amplification in some cycles might not occur, and therefore bands remain fainter.


The chance of these kinds of bands being sensitive to reaction conditions of course would be higher than those with higher intensity amplified with primers perfectly matching the priming sites. The most important factor for reproducibility of the RAPD profile has been found to be the result of inadequately prepared template DNA.

Term Paper # 4. Limitations of RAPD:

1. Nearly all RAPD markers are dominant, i.e., it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). Co-dominant RAPD markers, observed as different-sized DNA segments amplified from the same locus, are detected only rarely.

2. PCR is an enzymatic reaction, therefore the quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is laboratory dependent and needs carefully developed laboratory protocols to be reproducible.

3. Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

RAPD markers have found a wide range of applications in gene mapping, population genetics, molecular evolutionary genetics and plant and animal breeding. This is mainly due to the speed, cost and efficiency of the RAPD technique to generate large numbers of markers in a short period compared with previous methods. Therefore, RAPD technique can be performed in a moderate laboratory for most of its applications. Despite the reproducibility problem, the RAPD method will probably be important as long as other DNA-based techniques remain unavailable in terms of cost, time and labor.