Get in Touch with the Cutting Edge - Gene Chip Technology
Frank Döring, Ph.D.
Institut für Ernährungswissenschaften, Molecular Nutrition Unit, Technische Universität, München, Germany
phone +49-8161-71-3997, fax +49-8161-71-3999, email: doering@wzw.tum.de
Learning Objectives
 To understand the basic principle of the gene chip technology
 To enumerate basic applications of the gene chip technology
 To list potential applications of the gene chip technology in nutritional science
Background: Traditional Methods
In order to understand the basic principle of the gene chip technology, it may be helpful to
start with a description of a traditional method in molecular biology.
One example of such a method is called Northern Blot. The Northern Blot ist the most
common method for studying messenger ribonucleic acid (mRNA) level. There are similar
types of assays to detect and quantify DNA fragments (Southern Blot), proteins (Western
Blot) and protein-nucleic acid binding (North-Western Blot). By measuring mRNA levels
within a cell, one can indirectly measure the genes that are being transcribed and, by inference, the proteins that the cell is trying to make. In the Northern Blot, the mRNA from the
cell is isolated and purified from the biological specimen. The sample of mRNA contains
virtually all of the mRNA species from the specimen. It is then loaded onto a gel and the
various mRNA species are separated electrophoretically. The gel is then transferred onto
a membrane and is now referred to as a blot. Separately, a specific sequence of DNA is
created using a variety of techniques such as chemical synthesis from the genetic sequence (Oligonucleotides). This sample ist labeled by inserting tagged nucleotides (e. g.
32P, Cyanine-3) into the sequence. The labeled sequence is now referred to as the probe.
The probe is hybridized with the blot. Any place where the sequence of the probe aligns
with the sequence of an mRNA, there will be hybridization, that is to say, the probe will
stick to the blot. After washing, the probe will appear only where there was specific hybridization and be absent where there was no matching mRNA. By quantifying the amount of
tag on the blot, the relative amount of mRNA can be measured.
Principles of the Gene Chip Technology
The Northern Blot assay is an essential tool to molecular biologists. However, the problem
with this blot is that it is limited in the number of samples that can be analyzed simultaneously. If we take the simple case of the Northern Blot and extrapolate it to all of the genes
in the Human Genome (~ 30.000) and all of the experiments that would be required to assay an array of nutrients or pharmacologically active compounds, the number of assays
are required is overwhelming.
Therefore, a different approach with a significantly higher capacity to run assays was required. This aim was accomplished by novel technology allowing to perform many assays
in parallel rather than performing a long series of experiments sequentially. One of these
methods is the gene chip technology. This technology is fundamentally a parallel array
modified Northern blots.
The prototypic example of the gene chip technology was created in the Brown and Botstein laboratories at Stanford, USA. In this chip system, the probes are not labeled. In-
stead, they are attached to a membrane or to a glass slide. The probes are laid out in an
orderly array with a device called an arrayer. Basically, the probes (i.g. oligonucleotides)
are created and stored in microtiter plates. An arrayer removes a small amount of solution
from each well (a few nanoliters) and spots it onto the membrane or glass slide. Up to ~
105/cm2 different probes can be spotted on a glass slide. Separately, mRNA is isolated
from the experimental sample and a control sample. The mRNA from each sample is labeled with a tagged nucleotide. Normally, experimental and control mRNA are labeled with
two different fluorochromes that can be spectrally detected independently. The mRNA
from the samples is referred to as the target. Both target and control samples are hybridized against the spotted probes. If the experimental condition caused increased transcription of a given sequence, then there will be an excess of that color relative to the control
that is detected as an experimental:control ratio of > 1.0. If the experimental condition
caused decreased transcription of a given sequence, the experimental:control ratio will be
< 1. If the experimental condition had no affect, the ratio will be 1.0.
Another gene chip system has been developed by Affymetrix. In this system, instead of
arraying the spots, the spots are synthesized by a series of photo-activated steps with the
synthesis being directed by a series of photolithographic masks.
The third type of a gene chip was developed by Nanogen. Most molecules are electrically
charged and Nanogen takes advantage of this fact to help researchers quickly and efficiently analyze molecules like deoxyribonucleic acid (DNA). The technologies from Affymetrix and Nanogen can be also used to measure single nucleotide polymorphisms (SNP).
Applications of the Gene Chip Technology in Nutrition Research
For the field of nutrition to adequately harness the powerful tool of the gene chip technology, it is imperative that collaborations be formed with mathematicians and computer scientists. With the proper design and implementation of gene chip experiments, this approach
will provide the basis to understand how nutrients affect gene expression. By capturing
genetic pathways in computational form, the field of nutrition can augment the understanding of the metabolic pathways that are the benchmark of nutritional sciences. These genetic and metabolic pathways then can be manipulated through diet, environmental stimuli
and genetic targeting.
A recent example to study the influence of a nutrient on gene expression have been
achieved in a simple model system (Saccharomyces cerevisiae) with a well-defined genome. Lyons and coworkers used DNA microarray analysis to identify the yeast genes
involved in zinc homeostasis. Using gene chip technology, they observed that 15% of the
yeast genes were significantly altered by changing the zinc content of the media; on the
other hand the expression of 458 yeast genes is higher in zinc-deficient media.
A major advantage of using arrays, especially those that contain probes for the entire genome (such as the yeast chips used in this study), is that it is not necessary to generate a
hypothesis in order to select potentially important genes or mechanisms in advance. Instead of testing a hypothesis, a broader, more thorough and less biased view of the cellular response is obtained.
Another potential use of microarray technology in the field of nutrition is the „pharmacologic“ and „pharmacodynamic“ evaluation of nutrients in analogy to pharmacological or toxicological studies of drugs. By studying the cellular and molecular reaction to specific nutrients („nutraceuticals“ or „pharmaconutrients“) in defined doses, between population
groups and among individuals within those groups, one can begin to define the optimal
cellular doses to produce the desired molecular responses in a given subpopulation.
A second example for the application of the gene chip technology in nutritional science
was published recently by Weindruch and coworkers. To study effects of caloric restriction
on the gene expression profile of aging, caloric intake of mice was reduced to 76% of control animals. Age-related changes in gene expression profiles were remarkably attenuated
by caloric restriction Of the largest age-associated alterations 29% were completely prevented by caloric restriction and 34% were partially suppressed. Of the four major gene
classes (stress response, biosynthesis, protein metabolism and energy metabolism) that
displayed consistent age-associated alterations, 84% were either completely or partially
suppressed by caloric restriction. This striking effect of caloric restriction on age-related
changes in gene expression patterns supports the view that most of these reflect biological
age and can serve as biomarkers of aging. Also, these observations imply that some molecular aspects of the aging process are markedly suppressed by caloric restriction, suggesting that elucidation of molecular mechanisms will provide opportunities for intervention.
References

Hirschi KD, Kreps JA, Hirschi KK. Molecular approaches to studying nutrient metabolism and function: an array of
possibilities. J Nutr 2001; 131: 1605-1609

Roberts M, Geiger W, German JB. The revolution in microanalytic chemistry: a macro-opportunity for clinical nutrition. Am J Clin Nutr 2000; 71: 434-437

Lyons TJ, Gasch AP, Gaither LA, Botstein D, Brown PO, Eide DJ. Genome-wide characterization of the Zap1p zincresponsive regulon in yeast. Proc Natl Acad Sci USA 2000; 97: 7957-7962

Weindruch R, Kayo T, Lee CK, Prolla TA. Microarray profiling of gene expression in aging and its alteration by caloric restriction in mice. J Nutr 2001; 131: 918-923