Functional Genomics and DNA Chips

25 July, 1998

Functional Genomics and DNA Chips

Novel technical developments open up new fields and make for quantum progress in science. DNA chip is one of the recent examples.
As a post-genomics study, expression genetics of the genome dubbed as "functional genomics" has recently inspired the invention of several important techniques and their successful application for the analysis of differential gene expression in cells and tissues.

Techniques for studying mRNA by comparative and subtractive hybridization have been around for a long time. Differential display of mRNA by means of PCR (Liang P and Pardee AB, 1992: uid=92367009) is a new technique that made mRNA comparisons in eukaryotic cells relatively easy and serial analysis of gene expression method (Zhang L et al. 1997: uid=97301861) has identified ca. 500 transcripts expressed at significantly different levels among over 300,000 transcripts from ca 45,000 genes in normal cells and gastrointestinal tumors.

As complete sequences of the entire genome are being determined in a increasing number of organisms, the next step is the elucidation of the biological role of the genes. It is essential to be able to monitor the expression of a large number of genes in a quantitative and efficient way. DNA chip (GeneChip) technology comes in here with remarkable analytical power. Among the several DNA chip techniques reported recently, Affymetrix chip is the most noteworthy (Wodicka L et al. 1997: uid=98077736). The chip is very large-scale cDNA microarrays and produced as follows: Briefly, using conventional phosphoramidite-based DNA synthesis, a photolabile protection group on the terminal OH group of a spacer linked to glass substrate surface is removed by UV light shone through a photolithographic mask with aperture that could give a probe site (termed synthesis feature) of even 1 micrometer in size. In the next step, 5'-protected phosphoramidite is added to deprotected sites. After conventional treatments, a second mask is used to synthesize the next nucleotide at different arrays. It is possible to have an site density of 1 million/square centimeter. The process is repeated until the first nucleotide is synthesized at requisite probe sites and in Affymtrix chip, till 25-mer oligonucleotides are synthesized in situ in those sites. That is, 4 x 25-mer=100 chemical steps, 24 hours, 65,000 probe sites (50 x 50 micron in size) in 1.28 x 1.28 cm array. One array contains over 10 millions of a particular 25-mer oligonucleotide.

Since the complete sequence of S. cerevisiae genome is known, Affymetrix studied the expression of entire genes in minimal and rich media with the GeneChip. Twenty complementary 25-mers were chosen from 3'-end 1000 base sequence of each ORF (open reading frame, total 6218 in the yeast genome) based on the sequence information <http://genome-www.stanford.edu/Saccharomyces/>. Poly (A) mRNA was extracted, converted to cDNA and amplified in vitro 20-200 fold without significant bias and with fluorescent label. There were several careful controls such as a closely related mismatch partner probe (with a mismatch base substitution at the center of 25-mer oligo) in order to increase sensitivity and specificity. Expression level of mRNA from 0.1 to several 100 copies per cell was measured and nearly 90% of all yeast genes were expressed in minimal and rich media. Many previously uncharacterized genes were also observed. Similar DNA chip-based technology is now being extended to study gene expression characteristic of specific diseases (cancer, rheumatoid arthritis, human genetic diseases, etc).

For specific hitherto uncharacterized genes, another recent approach is the use of ESTs (expressed sequence tags) <ftp://ncbi.nlm.nih.gov/repository/dbEST> from a specific cell or organ. Vasmatzis G, et al (1998: uid=98081868) compared ESTs from a prostate cDNA library with those from other prostate libraries (normal and tumor) and from non-prostatic libraries and classified clusters of ESTs with high homology (BLASTn S=300; V=300; B=300; n=-20). When ESTs with no homology with those from non-prostatic libraries and without putative IDs (known enzymatic and other functions) were examined in RNA hybridization, 3 turned out to be prostate-specific.

The profile of gene expression is also used in drug discovery. NCI now possesses a panel of 60 human tumor cell lines representing leukemia, melanoma, and cancers of the lung, breast, colon, kidney, ovary, prostate, and CNS and the cytotoxic information of 49,000 compounds are stored in drug screening database. The patterns of cytotoxicity of an unknown compound may be compared in the computer analysis program COMPARE. The attempt was made to identify inhibitors of EGF receptor and c-erbB2 (Wosikowski K et al. 1997: uid=97477284). Their mRNA expression in the 60 cell lines was examined and the inhibition of mRNA expression by test compounds correlated with the growth inhibition of target cells. Fourteen compounds were thus identified as inhibitors, B4 being the most potent that inhibited autophosphorization of erbB2.

Until recently, the identification of unknown genes by homology comparison was of major interest for drug discovery clues among R&D scientists in the pharmaceutical industry. The pitfall here is that there are numerous genes whose mutations cause diseases and it is a difficult endeavor to establish the biological functions of new genes. Alternatively as discussed above, comparative mRNA expression under a specific disease condition may better lead to the discovery of genes whose functions are most likely unknown but directly involved in the disease occurrence. The conceptual change might be required for drug hunting in the age of functional genomics.　

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