DNA Fingerprinting: A Introduction
DNA is located in the cell nucleus and called as nuclear DNA. A small amount of DNA can be found in the mitochondria, where it is called as mitochondrial DNA. DNA has the ability to replicate and make copies of itself. The double-helix structure of DNA was presented by James Watson and Francis Crick in April 1953. They won a Nobel Prize in Physiology or Medicine for their discovery in 1962.
DNA stores all the information with the help of four chemical bases; adenine(A), guanine(G), which are called purines and cytosine(C) and thymine(T), called as pyrimidines. These base pairs are attached to a sugar and a phosphate molecule. Thus, a base pair, sugar molecule and phosphate molecule form a nucleotide. The two long strands of nucleotides forming a spiral is called a double helix. This double helix looks similar to a ladder. The sequence in which these base pairs occur determine the formation of various traits and building of an organism.
DNA is also found in the plants. All living organisms use the same principle of storing information - DNA and RNA. DNA is unique to each individual like fingerprints. Thus, DNA can be mapped to reveal the genetic make up of an organism. The technique of DNA fingerprinting was discovered by geneticist Alec J. Jefferys in 1984. He was carrying out studies on the gene for myoglobin. DNA fingerprinting in plants is used for protection of the ecosystem, identifying marker traits, identification of gene diversity and variation and mutations. There are various methods for plant DNA fingerprinting like Restriction fragment length polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Amplified fragment length polymorphism (AFLP) and Simple Sequence Repeats (SSRs).
DNA fingerprinting in plants involves the extraction of DNA from plant cells, quantification and quality assessment. While carrying out polymerase chain reaction (PCR) based duplication of DNA in RAPD, ISSR or SSR, diluted DNA is mixed with a master mix. The master mix contains PCR buffer, DNTPS, primer, water and Taq polymerase enzyme in a PCR eppendorf tube. The PCR machine is pre-programmed for the number of cycles, the DNA mixture is loaded and the cycle is carried out. After the cycle is completed, electrophoresis of the samples is carried out. AGE or PAGE electrophoresis can be used depending upon the technique. The samples are stained to reveal the banding patterns. After DNA has been isolated and enough copies of DNA are replicated, various methods explained below are used for DNA fingerprinting.
Restriction Fragment Length Polymorphisms (RFLPs)
In this method, unequal lengths of DNA fragments are obtained by cutting Variable Number of Tandem Repeat (VNTRs) sequences up to 30 sequences long with restriction enzymes at specific sites. There are different VNTRs, as there are different plant species, number and location of restriction enzyme-recognition sites. PCR amplification of DNA is not required for this method. The routine southern blot experiment can be used. The complimentary DNA sequences are radiolabeled on agarose gel for visualization in this method. This method is used to identify the origins of a particular plant species. This method is not much favored for DNA fingerprinting, as it has many drawbacks. The results cannot indicate the chance of match between two organisms. The other drawback of RFLPs is a costly process which involves lot of labor and money.
Randomly Amplified Polymorphic DNAs (RAPDs)
This method is most commonly used for primary assay. This method helps in screening the differences in DNA sequences of two species of plants. This method is used to search the sequences required for random amplification. In this method, using short single primers at low annealing tempratures, DNA is cut and amplified. Using electrophoresis and superimposing the gels, a banding pattern is identified. The gel is cut where the target band is found and the DNA is isolated and sequenced. This target is used to assess DNA from other cultivars. This technique is more cost-effective than RFLPs. The drawback for this method is that RAPDs lack specificity due to low annealing temperatures and easier reaction conditions.
Simple Sequence Repeats (SSR)
Simple sequence repeats are microsarellites. They show high degree of polymorphism. They are isolated using hybridized probes followed by their sequences. They are detected by gel electrophoresis using specific dyes or radiolabelling. The advantage of SSRs is that the amount of DNA required is less than RFLPs. The assays involving SSRs are more robust, making them more efficient than RAPDs. The drawback of this method is that seperate SSR primers are needed for each species.
Amplified Fragment Length Polymorphism (AFLP)
This method is a PCR based derivative of RFLP. Here sequences are selectively amplified using the primers. This method is more useful than RFLP or RAPD as more loci can be evaluated. AFLP helps in determining a large number of polymorphism. This method is also cost effective.
The advantages of DNA fingerprinting in plants are as follows:
â€¢ DNA fingerprinting is used for the identification of genetic diversity within a breeding population. It is used to identify a gene of interest. In the United States, it is also used to detect a genetically modified organisms in agriculture.
â€¢ RFLP markers are used to detect the genetic distance in wheat.
â€¢ RAPD markers are used for characterization, estimation of genetic relatedness and determination of genetic diversity of tea germplasm. It is also used to find genetic relatedness and difference in figs.
â€¢ AFLP markers help in assessing genetic diversity among cultivars such as wheat. It also helps detect higher level of polymorphism.
â€¢ DNA fingerprinting of herbal drugs can be useful in authenticating the various claims of medical uses related to the plants.
DNA fingerprinting has come a long way from being just a forensic tool. In plants, it has helped in identifying the plant genomics. It is useful in plant breeding. Many endangered plant species can be kept alive with the help of DNA fingerprinting. It is the effective tool for conserving the eco-diversity of our planet.
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