|
 |
 |
|
|
|
 |
|
|
3. Robot spotting fabrication of DNA microarrays with pre-synthesized probes (Stanford type microarray)
As the Human Genome Project continued toward its goal of characterizing the genomes of human and selected model organisms, and more and more information about genome sequence was accumulating, it became clear that new and much larger scale analytical methods were necessary to analyze gene differences between species and the functions of newly identified genes. High-density DNA microarrays were known for large scale DNA analysis11, 12), but the demand for inexpensive custom microarrays was mounting from researchers who were studying genes in different fields. Brown seized this opportunity and led his research group to develop the technology for fabricating inexpensive DNA microarrays suitable for custom preparation. In 1995, he succeeded in fabricating cDNA microarrays using a robot spotter17) and reported the first application of cDNA microarray to the gene expression studies18).
The novelties in their technology for the fabrication of DNA microarrays are the uses of high precision, high-speed robot spotter and cDNA or pre-synthesized oligonucleotides as probes. The use of the high precision, high-speed robot spotter allows researchers to prepare high-density microarrays on a very small area of solid support with good reproducibility. The use of cDNA or pre-synthesized oligonucleotides as probes gives researchers flexibility to prepare their own microarrays with their choice of probe content. Brown and his group developed a robotic spotting machine for arraying, and, in 1996, released information as to know-how, tools, and the design for the fabrication of DNA microarrays on the Internet19, 20). This disclosure of information for the self-fabrication of DNA microarrays has prompted the wide-spread use of DNA microarrays with pre-synthesized probes. It also paved ways for many companies to develop their own DNA microarrays with pre-synthesized probes based on their own technologies such as ink jet and spotting methods, and let them to enter to DNA microarray market.
Figure 2 shows a diagram for the fabrication of DNA microarrays with pre-synthesized probes using a robot spotter. Microarrays are fabricated on poly-L-lysine-coated microscope slides with a custom-built spotting machine fitted with printing tips. The tips are loaded with probes from 96-well microtiter plates and deposit small amounts of probe on the surface of the slides. After hybridization, the microarrays are scanned with a laser fluorescent scanner.
Since DNA microarrays can identify numerous genes at the same time, they are mostly used in the fields of gene expression and genetic analyses instead of being used as sequencers. Brown and his co-workers reported a quantitative analytical method for gene expression by conducting co-hybridization on a DNA microarray18, 21). Figure 3 shows a process diagram of gene expression analysis on DNA microarrays. Messenger RNA extracted from target tissue is labeled with green fluorescent dye and mRNA from reference tissue is labeled with red fluorescent dye. They are co-hybridized on a DNA microarray with cDNA as probes. The DNA microarray is scanned with a laser and the fluorescent patterns are memorized in a computer. The patterns are superimposed on the computer screen and analyzed. If the quantities of both mRNA are the same, the overlapping part turns yellow. If mRNA in the target tissue is more abundant in the reference tissue, the overlapping part turns green, and will turn red in the opposite situation. This way, researchers can tell which genes are activated or suppressed in target tissue. This method enables researchers to conduct quantitative gene expression studies and has become an indispensable method in genetic and pathological studies.
|
|
|
