Introduction

DNA Microarrays are a relatively new technology that allow researchers to monitor the RNA expression levels for thousands of genes at one time [1,2]. The underlying technique of hybridization to a known sequence has been used for quite some time in techniques such as Southern and Northern blots. DNA Microarrays have extended this basic technique by using much smaller amounts of DNA probe, and more importantly by allowing researchers to perform tens of thousands of hybridization experiments in parallel. This allows researchers to view the response of whole genomes to various stimuli.

The actual mechanics of making and using DNA microarrays is non-trivial. In order to construct microarrays with thousands of probes on a standard microscope slide it is necessary to place each spot only 200 microns away from it's nearest neighbor. To accomplish this high density robotics are used [3]. In general a robot is used to spot many different DNA molecules, which will be the probes for the microarray onto pre-treated glass microscope slides. The DNA is then covalently bound to the slide.

Now that the microarray has been constructed it can be used for a hybridization. The general protocol for performing hybridizations is as follows:

1.

RNA is extracted from an experimental state of interest, e.g. a strain of yeast with a gene deletion. RNA is also extracted from a control or reference state, e.g. a wild-type strain of yeast.

2.

The RNA from each strain is reverse transcribed to form a cDNA from the original RNA, this is referred to as the target. The cDNA from the experimental and the reference state are labeled with different fluorescent dyes.

3.

The cDNA targets are mixed together and are hybridized to the microarray. After hybridization the microarray is washed to remove non-specific binding.

4.

The microarray is then scanned using essentially a modified fluorescent microscope with a photomulitplier tube attached. This translates the intensity of each fluor hybridized to the microarray into separate 16 bit tiff files.

5.

The pixel intensities in the resulting tiff files are then compared to determine which RNAs are increased and decreased in the experimental vs. the control states.

Usually the DNA probes spotted onto the microarray are double stranded PCR products. Some of the first microarrays produced contained probes for every gene in yeast. These probes had been produced by PCR from genomic DNA using gene specific primers [1]. This approach would not work for higher eukaryotic organisms such as C. elegans, mouse, and human as these genomes contain many more introns than yeast. In order to produce DNA probes for these genomes researchers turned to Expressed Sequence Tags (ESTs) [2]. This technique has the drawback that the ESTs available do not cover all of the genes in these organisms. Also, it is very difficult to produce probes using PCR that can distinguish between differentially spliced mRNAs and to find SNPs.

Recently it has become possible both financially and chemically to bind oligonucleotides to microarrays. Although Affymetrix has been producing oligonucleotide microarrays for some time [4] the chemistry used is too expensive for most research labs. Technical benefits of using oligonucleotides include not having to perform PCR to create probes, and the fact that researchers are no longer dependent on the EST libraries for probes. With oligonucleotides it is possible to go directly from a genome sequence to creation of a probe. There are also functional advantages to using DNA oligonucleotides for probes. It is possible to design probes that can distinguish between differentially spliced mRNAs [5], probes that distinguish between highly related sequences [4], and also probes that detect SNPs.

Although oligonucleotides offer greater technical and functional benefits it is first necessary to design probes that can fulfill these objectives. While cDNA probes are imprecise they do contain a lot of sequence. Oligonucleotides probes are much smaller and it is important to avoid sequences that will be complementary to more than one gene, that will have secondary structure, and sequences with binding energies that are too high or too low. The objective of this work is to develop an initial program for use at UC Santa Cruz to create a yeast microarray that has features designed for investigating the regulation and impact of splicing on the cell.

Last updated on 7 December 1999 Maintained by sugnet@cse.ucsc.edu