2. Species-Specific Detection Using DNA Microarray-Based Techniques
(Discrimination Between Tuna species)
There are seven species and one sub-species of tunas in the genus Thunnus South.
The blackfin tuna T. atlanticus and longtail tuna T. tonggol are confined to
the Atlantic and Indo-Pacific, respectively. The southern bluefin tuna T. maccoyii
occurs only in the southern hemisphere, while the Atlantic northern bluefin
tuna T. thynnus thynnus and the Pacific sub-species T. t. orientalis are distributed
mainly in the northern hemisphere of the Atlantic and Pacific, respectively.
The Atlantic and Pacific populations of the northern bluefin tunas are well
isolated geographically from one another, as they are rare in the Indian Ocean
and in the southern hemisphere generally. The other three species, albacore
T. alalunga, bigeye T. obesus and yellowfin T. albacares tunas are considered
worldwide panmictic species.
Exact identification of the species and origin of marine products is necessary, particularly for foreign trade. The northern bluefin tuna (Thunnus thynnus) is most highly rated as a gsashimih product for the Japanese market, which may consequently have accelerated illegal fishing and trading irrespective of the decreasing stocks. Currently, the fishing season and amount of catch and trading in the Atlantic subspecies (T. thynnus thynnus) are severely regulated. However, discrimination of this species from the Pacific subspecies (T. thynnus orientalis) is nearly impossible when the few diagnostic external and internal morphological characteristics are removed, or when they are filleted. Therefore, genetic species identification should be useful.
A DNA fragment (ATCO) flanking the mitochondrial ATPase and cytochrome oxidase subunit III were employed for distinction of differences in the restriction profiles and nucleotide sequences between Atlantic and Pacific northern bluefin tunas. The entire nucleotide sequences of the ATCO fragments of these two subspecies to find DNA sequences were performed in the design of specific DNA probes (1). A magnetic-capture hybridization technique employing bacterial magnetic particles (BMPs: see Magnetic Bacteria for more detail), which are isolated from Magnetospirillum magneticum AMB-1, was applied to discriminate between Atlantic and Pacific subspecies of the northern bluefin tuna (Thunnus thynnus) using specific DNA sequences. The BMPs were magnetically concentrated, spotted in 100-ƒÊm-size microwell on microarray, and the signal detection was performed (Figure 1). This system employing DNA on BMPs may be useful for discrimination of these two subspecies by recognizing a single-nucleotide difference (2).
(Species-Specific Detection of Cyanobacteria)
Identification and detection of microorganisms in aquatic environments is important
in environmental monitoring, clinical, and food industrial fields. Methods based
on colony formation have been employed for the detection of viable cells in
microbiology. However, colony formation method takes 12-24 h even in the case
of bacteria and 1-2 weeks for assessing the microalgal cell viability. In addition,
the colony count method often underestimates the cell number since some microorganisms
isolated from sea cannot form detectable colonies.
Recently, the use of molecular techniques, such as PCR amplification, enables investigation of the distribution of microorganisms in aquatic environments. DNA-based sensing is useful to describe the existence of such microorganisms in environmental samples without requiring formation of their colonies. The bloom-forming genera of cyanobacteria (see Marine Biotechnology for more detail), Microcystis spp, produce toxins including hepatotoxic microcystins that are potentially health hazards for livestock and humans. Detection of the toxin-producing cyanobacteria of Anabaena, Nostoc, Microcystis, and Oscillatoria genera was investigated using an oligonucleotide probe. Genus specific oligonucleotide probes for the detection of Anabaena spp., Microcystis spp., Nostoc spp., Oscillatoria spp., and Synechococcus spp. were designed from the variable region of the cyanobacterial 16S rDNA of 148 strains (3). These oligonucleotide probes were immobilized on BMPs via streptavidin-biotin conjugation and employed for magnetic-capture hybridization against digoxigenin-labeled cyanobacterial 16S rDNA. The BMPs were spotted in 100-ƒÊm-size microwell on microarray (Figure 2). This work details the development of an automated technique for the magnetic isolation, the concentration of hybridized DNA, and the detection of specific target DNA on microarray. The entire process of hybridization and detection was automatically performed using a magnetic separation robot and all five cyanobacterial genera were successfully discriminated (4, 5).
3. Genome Analysis System Using On-Chip Device
A basic technique for DNA analysis in molecular biology will be divided with
four main processes, i.e. DNA extraction, DNA amplification, purification of
amplified products, detection of purified products (Figure 3). Automated systems
for DNA extraction, thermal cycler for DNA amplification and electrophoresis
for purification are commercially available. Lab-On-A-Chip technology based
on nanotechnology will lead to perform above entire process on a card-sized
device. Especially, we have focused on DNA extraction and amplification on a
chip.
(DNA Extraction from Whole Blood Based on Electrostatic Interactions)
A cascading hyperbranched polyamidoamine dendrimer was synthesized on the surface
of BMPs to allow enhanced extraction of DNA from fluid suspensions (Figure 4)
(6, 7). Characterization of the synthesis revealed linear doubling of the surface
amine charge from generations one through five starting with an amino silane
initiator. The dendrimer modified magnetic particles have been used to carry
out magnetic separation of DNA. Binding and release efficiencies increased with
the number of generations and those of bacterial magnetite modified with six
generation dendrimer were 7 and 11 times respectively as many as those of BMPs
modified with only amino silane. The binding will be due to the electrostatic
interactions between positively charged dendrimer modified magnetic particles
and DNA. The extraction method enables to purify 33 ng genomic DNA form 1 ?L
blood. Furthermore, this technique was fully automated using newly developed
liquid handling robots and bacterial magnetic particles (8).
(DNA Amplification on Micro-Chip)
Based on above techniques, a micro-chip for DNA extraction and amplification
was constructed (Figure 5). Amine-coated micro-chip was constructed for DNA
binding assay. The binding capacity reached to 6 ng per micro-chip. Furthermore,
a DNA amplification of alcohol dehydrogenase 2 (ALDH2) gene (176 bp) was successfully
achieved in micro-chip that the reaction volume was 0.5 ?L. The entire process
of DNA extraction and DNA amplification will be subsequently performed on the
micro-chip in future works.
4. Future vision
Our researches on DNA chip are based on nanotechnology, which has potentials
to yield new markets in various fields. Especially, gNanobiotechnologyh covers
a wide scientific area relevant to physics, engineering, biology and medicine.
The study of gNanobiotechnologyh brings forth a new interdisciplinary field.
In fact, recently, we have launched researches about cell assembling and cell-chip
based on gNanobiotechnologyh.
5. References
1. Takeyama H, Chow S, Tsuzuki H, Matsunaga T. Mitochondrial DNA sequence variation
within and between Thunnus tuna species and its application to species identification.
J Fish Biol 2001;58:1646-1657.
2. Takeyama H, Tsuzuki H, Chow S, Nakayama H, Matsunaga T. Discrimination between
atlantic and pacific subspecies of northern bluefin tuna (Thunnus thynnus) by
magnetic-capture hybridization using bacterial magnetic particles. Mar Biotechnol
2000;2:309-313.
3. Matsunaga T, Okochi M, Nakayama H. Construction of an automated DNA detection
system for manipulation of Microcystis spp. using specific DNA probe immobilized
on the magnetic particles. Electrochim Acta 1999;44:3779-3784.
4. Matsunaga T, Nakayama H, Okochi M. Fluorescent detection of cyanobacterial
DNA using bacterial magnetic particles on a MAG-Microarray. Biotechnol Bioeng
2001;73:400-405.
5. Matsunaga T, Takeyama H, Nakayama H. 16S rRNA-targeted identification of
cyanobacterial genera using oligonucleotide-probes immobilized on bacterial
magnetic particles. J Appl Phycol 2001;13:389-394.
6. Yoza B, Arakaki A, Matsunaga T. DNA extraction using bacterial magnetic particles
modified with hyperbranched polyamidoamine dendrimer. J Biotechnol 2003;101:219-28.
7. Yoza B, Matsumoto M, Matsunaga T. DNA extraction using modified bacterial
magnetic particles in the presence of amino silane compound. J Biotechnol 2002;94:217-24.
8. Yoza B, Arakaki A, Maruyama K, Takeyama H, Matsunaga T. Fully automated DNA
extraction from blood using magnetic particles modified with hyperbranched polyamidoamine
dendrimer. J Biosci Bioeng 2003;95:21-26.