Prof. Joe Cummins
Two kinds of techniques, DNA fingerprints and DNA barcodes, have revolutionized the identification of individual organisms and species. The use of these techniques does not alter the DNA of the organism and does not involve genetic engineering.
DNA fingerprints have been used to identify individuals in criminal cases, cases of disputed parentage and victims or warfare or accidents. DNA fingerprints are also used for identifying pathogens including viruses, bacteria and parasites. Individual plants, animals, fungus or alga and their progeny may be traced using DNA fingerprints.
DNA barcodes, on the other hand, use short DNA sequences that are present in all plants, animals, microbes or viruses, in order to identify individual species. To be useful the sequences are derived from genes that evolve rapidly (but not too rapidly) providing clear differences between species as they evolve. Ideally, one gene sequence would be used to identify species in all of the taxa (taxonomic groups) from viruses to plants and animals. However, that ideal gene has not yet been found, so different barcode DNA sequences are used for animals, plants, microbes and viruses.
A DNA sequence has been found in a mitochondrial gene inherited mainly through the maternal line, which effectively discriminates between most of the animal species. A segment of the cytochrome C oxidase gene 650 bases long has been elevated to the status of “the barcode of life” even though it is only effective in identifying animal species. Nonetheless, the entire biodiversity of life on earth has been targeted for barcoding and within the current decade [1, 2]
Recently, a 100-base fragment of the original barcode was found to be effective in identifying archival specimens and potentially useful for all taxa of the eukaryotes (organisms with nucleus in the cell) [3]. However, mitochondria and nuclear genes have different rates of evolution (with the former normally evolving faster), so estimates of biodiversity based on mitochondrial DNA may not be truly representative [4]. Furthermore, even though most animal species can be identified from the standard barcode, the cytochrome gene evolves too slowly in corals and sponges for it to be used as barcode identification in those species [5, 6]. Bearing in mind those caveats, the animal barcode based on a single DNA sequence works sufficiently well for most general purposes.
DNA barcodes in plants proved more elusive than those in animals. Plant mitochondrial genes are unsatisfactory while several potential candidates have been found in the chloroplast genome. Of these, the gene maturase K (matK) appears to provide the most reliable barcode, and was used to resolve the flora of biodiversity hot spots [7]. The matK barcode discriminated 90 percent of plant species [8]. MatK is nested in the group II intron of the chloroplast gene for transfer RNA lysine (trnK), and includes a domain for reverse transcriptase [9, 10]. Group II intron is a class of intron found in rRNA, tRNA, mRNA of organelles in fungi, plants, protists, and some mRNA in bacteria. Group II introns are self-splicing in vitro but employ maturase proteins in vivo. The use of two or more chloroplast barcodes has been advocated for the best discrimination in estimating biodiversity [11, 12], and impressive progress has been made in using chloroplast DNA barcodes for identifying plant species.
Tricholoma fungal pathogens have been discriminated using the mitochondrial ribosomal gene V9 sequence that displayed identical sequences within species but diverged between species [13]. Species identification in the fungi Trichoderma and Hypochrea was achieved using barcodes from the spacer regions of the nuclear ribosomal RNA genes [14].
The use of DNA barcodes is widespread in clinical microbiology and I am describing here only one of many applications. Very rapid detection of viral pathogens was achieved using a bio-barcode with a capillary DNA analyser, which allowed identification of the viral pathogen at very low levels within a few minutes [15]. An automated system described as a universal pathogen biosensor capable of identifying bacteria, viruses, fungi and protozoa was developed with funding from the United States Department of Defence. The technology uses mass spectrometer derived base-composition signatures obtained from PCR amplification of pathogen genomes to identify most organisms present in an environmental or tissue sample. The system uses computer software to handle the mass of information. The instrumental package called the Ibis Universal Biosensor is an automated platform for pathogen identification and strain typing [16].
In contrast to the DNA barcode which tags species, the fingerprint is designed to identify individuals or clones. DNA fingerprinting was invented by Professor Sir Alec Jeffreys of Leicester University in 1984. The first applications of the technique were in the identification of criminals and in paternity and immigration disputes. The method soon spread to the study of genetic lineages of wild and domestic animals and plants.
As described by Sir Alec [17]: “The most prevalent method of DNA fingerprinting used today is based on the polymerase chain reaction and analyses variation at short tandem repeat regions of DNA, also known as microsatellites. These highly polymorphic regions have short repeated sequences of DNA (the most common is four bases repeated, but there are other lengths in use, such as three and five bases). Because different people have different numbers of repeat units, these regions of DNA can again be used to discern between individuals. These repeat locations are targeted with sequence-specific primers and are amplified. The DNA fragments that result are then separated and detected using electrophoresis; instead of using radioactive probes, the gel is scanned directly and the DNA profile uploaded directly into a computer. Each variant displayed at a short tandem repeat region is usually quite common in humans. However, when looking at multiple regions, it is the unique combination of these variants in an individual that makes this method highly discriminating as an identification tool. The more repeat regions that are tested in an individual, the more discriminating the test becomes.” There is also a useful discussion of DNA finger printing on Wikipedia [18].
There are numerous examples of the use of DNA fingerprinting in agriculture, and I shall mention some of them. DNA fingerprinting traced the origin of a patented yellow bean variety Enola to an older Mexican variety, and Enola clearly lacked differences to distinguish it from the traditional Mexican cultivar [19]. DNA-based methods were used to identify grains in food mixtures, and to fingerprint grain varieties [20]. DNA fingerprinting identified the fruit tree species used in selection and introgression of new varieties [21]. DNA fingerprinting of microbial plant pathogens proved useful in diagnosis and disease management [22]. DNA fingerprinting of human pathogens such as the tuberculosis bacterium for over a decade has proven a powerful epidemiological tool [23]. The technique is valuable for establishing the lineages of animal breeding stock and perhaps even more so in identifying cloned animals and their progeny as they are offered for sale or export [24].
Article first published 16/10/08
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