Sir millions of copies of a given DNA
Sir Alec Jefferys first described DNA typing and fingerprinting in 1985. He detected some regions in human myoglobin gene sequence having some repeated sequences that were conserved (Jeffreys et. al., 1985). He also found that these sequences varied from person to person. Later, these repeated sequences were known as variable number of tandem repeats, or VNTRs (Nakamura et. al., 1987). Firstly Jefferys used RFLP (Restriction Fragment Length Polymorphism) using restriction endonuclease enzyme for DNA fingerprinting (Botstein et. al., 1980) but the technique was laborious and took large time to DNA fingerprint and required large amount of DNA (Butler, 2001). In 1980s, Saiki et. al. (1985) amplified VNTR loci by using Kary Mullis process of PCR. The technique was short, time saving, easy to use, required less DNA amount and thus easily replaced RFLP mapping of DNA typing (Butler, 2001).
PCR (Polymerase Chain Reaction) described by Kary Mullis is a process involving enzymatic amplification of short target sequences of DNA. The process is similarly modeled on natural DNA replication within a cell. PCR uses three steps namely denaturation, annealing and extension.
· Denaturation: First step of PCR involving heating of DNA sample to 94 degree at which double stand of DNA gets separated and becomes single stranded. Enzyme used in PCR i.e Taq polymerase can withstand this temperature and do not get denatured or deactivated.
· Sample is then cooled to annealing temperature of primer and primer bind with the target sequence.
· Temperature is further increased to 72 degree to facilitate the extension of primer according to the complimentary sequence of target sequence.
· These three steps run in loop or cycle and thus amplify the nucleotide sequence of interest.
This sequence is specified by 10 oligonucleotide primers in case of RAPD that are added to the reaction and are complementary to the 5′ and 3′ ends of the target DNA (Hartl and Jones, 2005). By performing 20-35 cycles of heating and cooling, millions of copies of a given DNA sequence can be produced (Fig.1)
Fig 1: Representation of a typical PCR amplification with known primers (Butler, 2001).
PCR is preferred over RFLP due to its simplicity, is time saving (Budowle et. al., 2002). Using PCR technique we can amplify an extremely small amount of DNA even if it is partially degraded because the procedure is sensitive. But due to being more sensitive result is very much affected by contamination (Budowle et. al., 2002).
In designing a typical PCR reaction, the sequence of the target area of DNA must be known. Knowing the nucleotide sequence of the target DNA allows primers to be synthesized that are complementary to the boundaries of the target sequence. The two primers added to a PCR reaction bind to the 3’end of each strand of DNA and serve as the initiation points for replication catalyzed by DNA polymerase (Hartl and Jones, 2005). Random Amplification of Polymorphic DNA, or RAPD, is a variation of traditional PCR and is designed to be used with DNA templates whose sequences are not known or well characterized. Williams et. al. (1990) used single oligonucleotide primers with arbitrary nucleotide sequences to amplify polymorphic DNA segments that are inherited in a Mendelian fashion and was able to construct genetic maps among members of plant and animal species. Because the primers used exhibited a high degree of sequence variability, polymorphic regions of the genome can be amplified in the absence of specific knowledge of nucleotide sequence (Williams et. al., 1990). In figure , primers labeled as 2 and 5 are able to produce a product, product A, and primers 3 and 6 also have opposite orientations and are in close enough proximity to produce product B. Primers 1 and 4 are also in opposite directions, but are not close enough to direct the efficient amplification of a PCR product.
Fig: RAPD analysis producing two products. This example shows a simple example of RAPD primers binding to a DNA template in an orientation where two different size products were produced.
Variation in the products produced with all PCR procedures occurs due to nucleotide sequences differences occurring in the DNA template. In fig, the DNA template is different from the figure shown above, because the primer labeled number 2 does not bind to DNA Template #2 and product A is not produced.
Fig: RAPD analysis producing one product. This picture shows a simple example of how a different DNA template has different binding patters and therefore, different products produced.
If amplification products from the templates shown in figures 5 and 6 were analyzed using gel electrophoresis, which separates DNA molecules based on size, results shown in fig., would be obtained.
Fig: Results of the simple RAPD example. Agarose gel results of the two DNA templates. Lane one contains a size ladder, while lane 2 portrays the products produced in DNA template#1 and lane 3 portraits the products produced from DNA template#2.
RAPD can be a powerful tool for gene mapping, population genetics, pedigree analysis, phylogenetic studies, surveying DNA for damage or mutation, and strain identification (Atienzaret. al. , 2001). In most studies, 10bp oligonucleotide primers with at least a 50% GC content seem to represent optimal primers for RAPD analysis (Williams et. al. , 1990). An example of the forensic use of RAPD occurred when an investigator needed to quickly determine if maggots found inside of a body bag were the same as the pupae found on the floor under the corpse (Benecke, 1998). There was not enough time to let the pupa grow, so DNA testing was needed. Results of RAPD analysis showed the arthropods being compared were genetically identical, and the testing was considered successful (Benecke, 1998). The advantages of RAPD are that only a small amount of template DNA is needed, a specific primer is not needed, the sequence of the genome of the organism does not need to be known, and the procedure is relatively easy, quick, and inexpensive.