DNA Chips

A corn chip???

If you work much in the field of biochemistry or molecular biology in the future, you will almost certainly come in contact with what people are now calling DNA chips. These are large arrays of DNA sequences attached to some surface which can be used in hybridization measurements where you want to follow the level of, for example, an mRNA for a large number of different genes all at the same time.

OK, let's back up a second. If you are studying the expression of a gene, or simply want to know which genes in an organism are being expressed under which conditions, you would often do the following. First, you would isolate the total mRNA from the organism under whatever conditions you wanted to measure gene expression. Then you would often generate what is called a cDNA library (the "c" stands for "copy") which is a DNA duplicate of the population of mRNAs that you just isolated. This is done with an enzyme known as "reverse transcriptase" which (without a primer) copies RNA back into DNA. People often like to work with DNA because it is less likely to be chewed up by enzymes (RNAase is very hard to get rid of). In this case, it is also easier to label, but more on that later. Now you have a set of DNA fragments whose sequence and concentrations reflect the mRNA population. Now let's say I had 10 genes I was interested in and I wanted to know if under the conditions I grew my cells these genes were highly expressed. I would probably have oligonucleotides made from the know sequences of those genes and using those as probes, I would hybridize them to the cDNA library. Usually in this case I would label the probes either fluorescently or radioactively so that I could tell which probes hybridized and to what extent.

OK fine, but along comes genomic sequencing. Now we don't just know the sequences of 10 or 20 genes in the genome, we know the sequences of thousands and thousands of genes in the genome. We would like to see how changing the environment of the cells changed to expression level of all of those genes. So, we want to make thousands and thousands of probes to hybridize to our cDNA library. OK, now we have a problem. First, it would be very expensive to have someone make thousands and thousands of individual probes for all the genes you wanted to look at. Second, it would take an immense amount of time. Third, even if you had them, it would take forever to do all the hybridizations and check all the gene levels. I mean, just how long do you really want to be a graduate student anyway?

What we need to be able to do is to make these probes all at once and then to use them all at once. This means doing things in parallel quickly and doing them on very small scales. Where have we all heard that song before? The electronics industry. So how do they make all those little tiny circuits that power the computer I am writing this on anyway? The process is called micro (or these days nano) fabrication and it involves placing a layer of some active stuff (like metal or semiconductor) on the surface of a material and then covering it with something call photoresist that can be polymerized (or de-polymerized) by light. Then you make a mask which determines where the light hits the photoresist. Then you shine light on the system and only polymerize the photoresist in a particular pattern. Now you wash off the unpolymerized stuff. Now you add some nasty acid or something that will eat away the metal or semiconductor layer underneath the photoresist, but only where the photoresist isn't. Know you add something else that disolves the photoresist. Now you are done with the first layer and you put down another layer of stuff and do this again. In this way you put down patterned layer after patterned layer of conductors, semiconductors, insulators, etc. until you have built up a function device -- Pentium III's use require like 500 such steps to generate them.

What does all that have to do with biochemistry? Well, back in the early 90's some clever folks said, why not do the same thing with DNA? In order to understand this, you need to understand how people make oligonucleotides. It is all done by solid phase synthesis these days. I won't go into the details of the chemistry, but you have a linking group on some surface (usually a bead in a column) and you attach the first base to that group. It has some blocking group on it so that you can't attach two bases at once. Next you remove the blocking group. Then you add another base of your choosing, etc. Layer by layer… sounds a bit like chip fabrication… hmmm. Now if we could only pattern it with light. What if the blocking groups were not removed chemically, but rather were photodissociated? Now if we built the DNA up on a flat surface, we could take the blocking groups off of only certain specific regions of the surface. One those regions would accept the next base. In this way, we can build whatever sequence we want anywhere on the surface as dictated by the light images we shine on the surface. In this way, people have build arrays of DNA probes on a "chip" with as many as 500,000 different probing sequences. Now, if we label our cDNA library with a fluorescent dye and then hybridize it to the chip, we can check the level of expression of every gene in the whole genome all at once by taking a picture of the chip with a CCD camera or something like that.

At this particular instance, these big chips are very expensive and only can be used once. They are also limited to about a million different sequences. However, this is all changing so fast that within a couple of years this will be a pretty cheap technology and one will be able to probe many millions of sequences at a time. Once the human genome is complete in a few years, they will also be able to look at your DNA sequence rapidly this way and tell you what genetic risk factors you have and devise (we hope) ways of avoiding those problems. Pretty cool stuff.

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