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Australasian Biotechnology, Vol. 12 No. 1, 2002, pp. 38-39

BIOTECHNOLOGY MINI REVIEW

DNA MICROARRAYS: MANUFACTURE AND APPLICATIONS

Mark Street

c/- Biotechnology Program, University of Queensland, Qld 4072 (mestreet76@hotmail.com)

Code Number: au02009

Abstract

DNA microarrays have greatly assisted scientists in their genomic endeavors. Microarrays are simply pieces of immobilized DNA sequences bound to a solid phase that has the ability to interact with a complementary piece of DNA. Whilst relatively simple in their design and manufacture, microarrays have multiple applications including genotyping, gene expression estimation as a diagnostic tool, drug selection and the identification of signature genes. Microarray technology is still in its infancy and is anticipated to advance rapidly in many aspects.

Introduction

The Human Genome Project has allowed considerable progress in the construction of physical and genetic maps and the identification of genes involved in human sickness (1). Indeed, the rapid sequencing of the entire genome of people, as well as that of other organisms, provides a wealth of information. In fact, the acquisition of this information brings to light a number of useful purposes such as complex genotyping, gene expression and the identification of ‘signature genes’. Much of this may be achieved with the use of DNA array technologies. The subject of this review will be to define DNA microarrays; the manufacture of DNA arrays; its applications, particularly in the diagnostic and pharmaceutical fields; and finally, to indicate the future directions of the technology.

What are DNA microarrays

DNA microarrays typically consist of thousands of immobilized DNA sequences that are bound to a surface. Arrays are used to analyse a sample for the presence of gene variations or mutations, or for patterns of gene expression. Both are based on the hybridization of the immobilized sequence with a sample that contains DNA that is of a complementary sequence. Shi (13) best describes DNA arrays as an orderly arrangement of samples. He goes on to say that arrays provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns.

The composition of I)NA on the arrays is of two general types. The first type involves the binding of oligonucleotides, of approximately 20-25 nucleotides in length, to a glass or nylon surface. These arrays are frequently used in genotyping. The second type involves the binding of complete or partial cDNA bases to a surface. These arrays are generally used for relarive gene expression analysis.

Manufacture of microarrays

There are several methods in manufacturing DNA microarrays, which may be categorized into one of three approaches (10). The first approach involves in-situ synthesis of olignucleotides or peptide nucleic acids (PNAs). Marshall and Hodgson (10) note that Affjymetrix uses this approach by using photolithography. This method involves the use of modified nucleotide phosphoramidites bearing a photo-labile protecting group, allowing mercury light to be used as the activating agent in the synthesis reaction (8). A single round of synthesis involves light-directed deprotection, resulting in the production of a 5 hydroxyl group capable of reacting with another nucleoside. Flooding the surface of the chip with one of the four bases results in the selective coupling of that base to each deprotected region on the synthesis surface. This process is repeated to produce an oligonucleotide probe. This method, whilst laborious, is considered to be the most effective strategy for generating very high-density arrays. Indeed, Lemieux (8) notes that this method currently allows approximately 250 000 features/cm² for 20 met oligonucleotides.

Whilst this method has many advantages, particularly pertaining to the large densities, there are also disadvantages. In particular, there is a significant amount of design work required and the costs associated with mask design are reasonably high (8). Another in-situ manufacturing technology is the ink-jet method, which is used by a number of companies such as Hewlett-Packard, Protogene and Incyte. This method involves the delivery of one of the four phosphorarnidites into hydrophilic wells that have been prepared photolithographically. The surface is then washed with oxidizer and deblocking agent to complete a round of coupling. Whilst the ink-jet technology arrays have a lesser density than the light-directed technology, it is far simpler to operate (8).

The second way of making arrays is by spotting. This method involves printing oligonucleotides or oligopeptides onto a glass support using direct touch or fine micropipetting. A good example of this is Majer’s microquill system (4). Its array pins are small enough to spot samples of approximately 75 microns and has the accuracy to deliver more than 400 spots, which is approximately equivalent to 0.3 to 0.6 nanolitres per spot. The pins are usually fitted into a robotic arm called an arrayer. The arm is controlled by software that allows the user to place genes in select areas and configurations on the glass slide. The pins, by capillary action, draw up a small amount of a solution containing the DNA for a single gene and deposits it in a precise location on a glass slide. The spotted genes are linked to the surface of the glass by either covalent bonds or charge interactions. The last way of making arrays is to ‘spot’ cDNAs directly onto the chip surface (10). The surfaces are fabricated with a positively charged coating, and cDNA fragments suspended in a denaturing solution are then printed directly onto the surface.

The second approach to cDNA mmcroarray fabrication utilizes the piez.oelectric method. This method involves a printer head that stops at each spot and dispenses a small drop, of one of the four bases, onto the coated surface. This technology has the potential to deliver 100 picoliter droplets at a rate of approximately 10 000 droplets/s (8), which in turn creates very high-density microarrays.

Microarray applications

DNA array technologies provide rapid and cost-effective methods of identifying gene expression and genetic variations. It is for these reasons that the arrays are currently being used, and will continue to be used, in a number of applications.

Firstly, DNA array technology provides a method for rapid genotyping, facilitating the diagnosis of diseases for which a gene mutation has been identified. Indeed, Ramsay (12) notes that array technology has given rise to polymorphism screening and mapping of genomic DNA clones for many diseases, such as hypertension and cancer. The latter has received a great deal of focus by research groups of late. There is evidence to suggest that single nucleotide polymorphisms (SNPs) from over 64, 000 probes have recently been analysed with the help of microarrays to detect small deletions and insertions in the BRCA1 and BRCA2 oncogenes (2).

Another application of DNA microarrays in the area of diagnostics is the analysis of gene expression. Gene expression is generally measured by the use of cDNA arrays. Typically, two probes are hybridized on a single array. The expression of a gene in an experimental situation is then expressed as a relative ratio with respect to the control sample (3). Gene expression studies have predominantly focused on the analysis of tumors and cancers. One such study involved the testing of gene expression variations of normal and abnormal oesophageal epithelium tissues (9). The pathological characteristics define five stages of progression, namely normal, dysplasia I (mild dysplasia), dysplasia II (moderate dysplasia), carcinoma in-situ (CIS) and squamous cell carcinoma of oesophagus (SCC). The experimentation utilized cDNA microarrays, which was confirmed by semi-quantitative reverse transcription polymerase chain reaction and immunohistochemistry. In analyzing the data, Lu (9) contends that cDNA microarray technology is a useful tool to discover genes frequently involved in oesophageal neoplasia and provides novel clues to diagnosis, early detection and intervention of SCC.

In addition to microarrays application to diagnostics, it may also be useful in pharmacology, particularly for drug selection, the discovery of therapeutic targets and for the prediction of drug or toxin activity. The abnormal expression of a gene involved in drug metabolism can manifest as an atypical response to therapy. With the use of microarrays, an individual's gene expression of a drug-regulated may be analysed and compared to the expression of another individual's gene. Kawanishi (6) gives the example of a study that involved the testing of gene expression of individuals administered atypical antipsychotic agents, such as clozapine, olanzapine and quetiapine. These drugs have been well received due to their fewer side effects. Kawanishi (6) notes that the response to even these drugs is heterogeneous, and psychiatrists usually prescribe antipsychotics based only on the clinical symptoms since they lack the biological evidence, which would help to make decisions of the appropriate drug for each individual patient. With the emergence of microarrays, however, the testing of these antipsychotic drugs efficacy by means of detecting an individual’s gene expression may provide clinicians with a patient profile and a guide to the suitability of these drugs.

In addition to drug selection, microarrays may also serve in the identification of signature genes indicative of a disease process, which can identify candidate targets for therapeutic intervention. In addition, arrays may also assist in the identification of genes that display altered expression in a given cell or tissue when exposed to a drug or toxin. There are several companies that have commercialized microarray chips for the detection of environmental and chemical toxins. One such company is the collaborative team of the Syngenta Central Toxicology Laboratory and AstraZeneca Pharmaceuticals. This team has recently designed and created a microarray named the ToxBlotll, which is the largest custom cDNA microarray available for toxicity characterization known (11).

Future prospects

Microarray technology is undoubtedly still in its infancy. It is anticipated that the technology will advance in several aspects. In particular, improvements in the automation of the fabrication of microarrays which will reduce the manufacturing costs and increase its throughput (8); a reduction in DNA spot size and advances in scanner technology which will allow greater densities; and the manufacture of chips for the analysis of other biomolecules. Already, there are several institutions and companies designing and manufacturing chips that are spotted with proteins instead of oligonucleotides or cDNA (7).

In addition, as our knowledge base of microarray expands, laboratories will have databases with expression profiles for hundreds, even thousands of genes (5). These gene expression databases are anticipated to complement the human genome sequence databases.

Conclusion

Microarrays have proven to yield a great number of applications to the field of science and health. Indeed, they provide rapid diagnosis of genetic diseases, facilitates drug selection, identifies candidate targets for therapeutics and has the ability to predict toxin or drug activity. The development of micloarrays utilizing DNA as well as other biomolecules, has the potential to revolutionise biotechnology.

References