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ISIS Report 16/10/08
DNA Identikit
Barcode for Biodiversity & Fingerprint for
Everything Else
Prof. Joe Cummins
A fully referenced
version of this article is posted on ISIS members’ website. Details
here
An electronic version of this report with full references can be downloaded
from the ISIS online store. Download Now
The identity of organisms and species
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.
DNA barcodes in animals
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.
Plant DNA barcodes
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.
DNA barcodes in microbes and viruses
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].
DNA Fingerprint
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].
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