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DNA-ARRAY CHIP TECHNOLOGY
 
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PART ONE: In vitro hybridization:

First we need to separate the two strands of "dsDNA" to reveal the bases hidden inside the helix. "ssDNA" is formed. The newly revealed bases can thus form H-bonds with any RNA's that happen to have perfect matches the sequence in the ssDNA. Here is how it is done.

The ssDNA and the RNA are mixed in the same 0.3M NaCl solution; heated to 67c for about 90 minutes and then cooled. RNA that is not hybridized is destroyed by addition of RNase, which only hydrolyzes ssRNA and not hybridized or dsRNA. All the big molecules are precipitated with trichloroacetic acid and filtered out. If the RNA is radioactive, then one finds radioactivity clinging to the DNA in a very specific manner due to complementary pairings.

PART TWO: Filter hybridization:

These are the types of nitrocellulose filters used above.

Dr. Agnar Nygaard, head of biology at the Univ. of Oslo Norway, was on leave and working in the lab of Dr. Ben Hall at the Univ. of Illinois in 1962, when he serendipitously discovered that ssDNA quantitatively adhered to nitrocellulose filters. (He forgot to add the trichloroacetic acid to his hybrid mixtures mentioned above. This immediately gave him the idea that perhaps the hybridization could be carried out on the filters rather than in aqueous solutions in test tubes. Now that the ssDNA is fixed to the filter, only a tweezers is needed to move the "DNA-filters" through the rinses and RNase steps. This shortened the procedure from 5 days of centrifuging, etc., to a single afternoon. What is more, a great many different DNA-filters could be manipulated in that one afternoon. It was estimated that this finding sped up hybridization studies more than 500-fold.

On the left, below, is a highly magnified view of a ssDNA strand stuck in places to the surface of the filter. It is not known what sort of bond connects the ssDNA to the nitrocellulose. On the right is a view of that ssDNA now annealed with a couple of sections of RNA to form the hybrid.

The way the above hybrid is formed and manipulated on the filters is extremely simple and straightforward: merely soak them in RNA solutions and heat them for 90 minutes at 67c. Next come a brief incubation or digestion with RNase and then a few rinses to eliminate all the digestion scraps.

Here are shown the fixing of two different ssDNA's to separate filters. (Colors are, of course, only for diagrammatic reasons.)

One of the first experiments to check this method for its specificity was to see if the ssDNA's isolated from two different organisms hybridized differently when soaked in various types of RNA's. Note here that filters of two different DNA's are in the same containers. One can place filters of many different DNA's into one container without any cross-reaction. This greatly contributed to the efficiency of this filter method over the previous in vitro methods.

PART THREE: Chip hybridization:

A "DNA-Chip" is, in a way, like a large collection of different, very small filters, each one with a different ssDNA stuck on it. Because of the complexity, these are generally produced commercially by a number of companies. Sometimes these ssDNA's are synthetic as they were polymerized or "constructed" base by base in vitro. Hence they are called "c-DNA". Alternatively, they might be ss-fragments of natural DNA's, such as from a variety of pathogens. Or they might be fragments of cut-up chromosomal genetic material.

To the right is an example of one way to begin deriving fragments from natural dsDNA. Here the chromosomal DNA has been treated with a restriction endonuclease, and then all the fragments are separated by the process of gel electrophoresis. Each of these fragments could then be isolated and then treated with another restriction endonuclease to yield even more fragments. Once several thousand different fragments are made, they can be placed and stuck one-by-one into the little squares of the "chip." Of course the reason that they stick is different than with the nitrocellulose filters, because the chip substrates are of completely different materials.
 
In summary, each of the hundreds of little squares on the chip has stuck a different piece of ssDNA.

A common way of using the DNA-array chips is for visualizing the different genetic expressions between pairs of cells. Examples may include a cancer cell and a normal cell, or a fetal cell and an adult cell, or the cells of different tissues, or even between people, one of whom is fighting a disease and the other is normal. Let's see how this works.

The CONTROL cell-type is allowed to metabolize normally. Then the RNA's are isolated from it. These RNA's will include the full spectrum of RNA types: rRNA, tRNA, and the many different mRNA's and the newly discovered
micro-RNA's (μ-RNA), which seem to have powerful regulatory functions. These control RNA's are then tagged covalently with a fluorescent molecule that glows green. Meanwhile, the cancer cell, for example, also has its RNA's isolated and they are tagged with a dye that fluoresces red. Then the two RNA's solutions are mixed and applied to the chip under hybridizing conditions not unlike those used long ago with the nitrocellulose filters. On the chip, the various RNA's compete for their complementary segments of ssDNA.

Four outcomes are possible:

Type of competition	Resulting Color(s)*
Neither can find a hybridization site	Black (no color)
Normal cell makes RNA but cancer cell does not.	GREEN
Both cells make those particular RNA's	BOTH COLORS
Cancer cell makes RNA but normal does not.	RED
The mechanical scanner does not blend colors like our eyes do, but rather measures the amount of each color.

Thus the overall hybridized chip is a kaleidoscope of colors. Wherever the cancer or other experimental type of cell is hyperactive, the squares are red and stand out rather sharply.

Now for a few quiz questions to see if you understand what is going on!

1. Suppose your two human cell types from which the RNA was isolated were identical, what color(s) would the chip glow? The cells would be either black (no activity in that genetic segment), or both red and green.
   
2. Suppose you made a mistake and used a chip with E.coli ssDNA on it rather than a chip with human ssDNA arrayed on it, and you applied human control and human cancer derived RNA's on it? Black.
   
3. You have a human ssDNA chip, and your RNA sources are those of a human male and human female. Why would you expect the chip to be mostly green? Why not completely green? Why some black? Why some red? You figure it out!
   
4. Forensics: You were fortunate to be able to gather some skin cells from under the fingernails of a murder victim and cultivate them to give you "crime scene" RNA, which you tagged to glow red. You then also made green RNA's from both the victim's own skin cells, and from a suspect's skin cells. You used two human ssDNA chips. On one you applied a mix of the crime scene and the victim's RNA, and on the other you applied crime scene and suspect's RNA. What would you expect to see if (1) the suspect was innocent, and (2) the suspect was guilty? You figure it out!
   

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DNA-CHIP MAKERS
Symbol	Name of Company
AFFX	Affymetrix
A	Agilent Technologies
AHM	Amersham Biosciences
unlisted	Axon Instruments
unlisted	BioDiscovery
unlisted	Clontech
GNSL	Genomic Solutions
unlisted	Mergen
(MOT)	Motorola Life Sciences
NGEN	Nanogen
unlisted	Partek
PKI	PerkinElmer
unlisted	Rosetta Inpharmatics
unlisted	Spotfire
VRK.TO	Virtek Vision International
PROTEIN-CHIP MAKERS
Symbol	Name of Company
BCOR	Biacore International AB
BSTE	Biosite Inc.
CIPH	Ciphergen Biosystems Inc
LSBC	Large Scale Biology Corp.

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