DNA Microchip Technology

What is a DNA microchip?

Scientists know that a mutation - or alteration - in a particular gene's DNA often results in a certain disease. However, it can be very difficult to develop a test to detect these mutations, because most large genes have many regions where mutations can occur. For example, researchers believe that mutations in the genes BRCA1 and BRCA2 cause as many as 60 percent of all cases of hereditary breast and ovarian cancers. But there is not one specific mutation responsible for all of these cases. Researchers have already discovered over 800 different mutations in BRCA1 alone.

The DNA microchip is a mew tool used to identify mutations in genes like BRCA1 and BRCA2. The chip, which consists of a small glass plate encased in plastic, is manufactured somewhat like a computer microchip. On the surface, each chip contains thousands of short, synthetic, single-stranded DNA sequences, which together add up to the normal gene in question.

What is a DNA microchip used for?

Because chip technology is still relatively new, it is currently only a research tool. Scientists use it to conduct large-scale population studies - for example, to determine how often individuals with a particular mutation actually develop breast cancer.

As we gain more insight into the mutations that underlie various diseases, researchers will likely produce new chips to help assess individual risks for developing different cancers as well as heart disease, diabetes and other diseases.

How does a DNA microchip work?

To determine whether an individual possesses a mutation for BRCA1 or BRCA2, a scientist first obtains a sample of DNA from the patient's blood as well as a control sample - one that does not contain a mutation in either gene.

The researcher then denatures the DNA in the samples - a process that separates the two complementary strands of DNA into single-stranded molecules. The next step is to cut the long strands of DNA into smaller, more manageable fragments and then to label each fragment by attaching a fluorescent dye. The individual's DNA is labeled with green dye and the control - or normal - DNA is labeled with red dye. Both sets of labeled DNA are then inserted into the chip and allowed to hybridize - or bind - to the synthetic BRCA1 or BRCA2 DNA on the chip. If the individual does not have a mutation for the gene, both the red and green samples will bind to the sequences on the chip.

If the individual does possess a mutation, the individual's DNA will not bind properly in the region where the mutation is located. The scientist can then examine this area more closely to confirm that a mutation is present.

Last Reviewed: November 3, 2008 （Source：National Human Genomic Program）

DNA Microchips

Perhaps the best-known type of surface attachment involves DNA oligonucleotide microarrays (so called gene chips). Their importance to the study of genetics was illustrated by the appearance in (1999) of an entire supplemental issue of Nature Genetics dedicated to the subject. These chips have been used to enable rapid sequencing of DNA, for the detection of RFLPs (Hacia & Collins, 1999) , for monitoring the presence of microorganisms in the food industry (Kuipers, 1999; Kuipers et al., 1999) , or the environment (Guschin et al., 1997; Oh & Liao, 2000) and to assay gene expression in a wide range of organisms (Harrington et al., 2000) . More recently, chips have been manufactured with a three-dimensional array based on the use of a polyacrylamide matrix (Guschin et al., 1997) . These are likely to lead to faster screening techniques and the use of larger “databases” of genes on the chips for expression studies. Microchip development has also spawned a vast collection of associated industries producing clone-sets, arrayers, scanners and software for data analysis (Epstein & Butlow, 2000) . It seems likely that single chips will get smaller, moving toward the nanoscale and the detection/use of single DNA molecules.

Currently, there are two major variants of DNA chip technology based on the type of immobilised DNA. In the first format, the immobilised probe consists of cDNA (500-5000bp DNA fragments produced by copying expressed genes) spotted onto a surface using robotics. The second format, consists of 20-25-mer oligonucleotides (or peptide nucleic acid - PNA) synthesised in situ (such as the gene chips produced by Affymetrix), or by conventional synthesis and then immobilised. The oligonucleotides are of known DNA sequence allowing detection of complementary sequences. Therefore, the DNA on the chip acts as a probe in a hybridisation reaction (Souteyrand et al., 2000; Southern et al., 1999) . The arrays come in various sizes from macroarrays to microarrays and beyond. Macroarrays are generally produced by hand, or using simple robots to pipette DNA solutions onto prepared surfaces and contains “tens to hundreds” of spots over an area of 500 mm or more. Microarrays contain many thousands of spots over an area of less than 500 mm.

Gene chips for cancer detection

DNA microchips have been used extensively for research purposes, but they also have a powerful use as a biosensor. For example, the onset of prostate cancer involves the differential expression of approximately 500 genes. A prototype chip for prostate cancer detection is already under development by the biotechnology firm Darwin Molecular (Bothell, Washington USA). They predict that within a year they will have a system that can distinguish slow-growing cancers that do not require immediate treatment and those aggressive tumours that require surgery (Zajtchuk, 1999).

Detection of hybridisation usually involves the use of PCR amplified, fluorescently-tagged DNA. Generally, for gene expression studies, this involves amplification of a pair of mRNA samples (i.e. a control and an experimental sample) using different fluorophores for each sample. Identical amounts of the amplified cDNA are hybridised to the same DNA chip and fluorescent intensities compared (Winzeler et al., 1999) . The use of two fluorophores overcomes problems associated with different amounts of probe DNA deposited at the specific spots on the chip.

The first consideration in the design of a DNA chip is the support material and the production of simple arrays in a research environment was discussed by Cheung et al. (1999) . Most usually, this involves the use of glass. However, recently silicon has also been employed as a support allowing the use of its semiconducting properties (Souteyrand et al., 2000) . Electrochemical deposition of oligonucleotides, via pyrrole groups is used to attach the DNA to the surface of electrodes (Livache et al., 1998a) . These electrodes can interact with the silicon allowing for the development of new techniques for detecting hybridisation based on the semiconductor properties of the silicon. A typical 128-sample grid is shown in Figure 17. The chip uses polarisation of the electrodes (Souteyrand et al., 2000) to prevent diffusion, allowing the samples to concentrate ensuring hybridisation occurs in a few seconds. After hybridisation, a short burst of the opposite polarisation will release weakly bound molecules improving signal-noise ratios. Finally, the charge on the DNA strands allows detection of hybridisation without using fluorophores, through the field-effects on the electrodes.

Perhaps the company most associated with DNA microchips, Affymetrix, believes that the capacity of DNA microchips is following Moore’s Law, which predicts a two-fold increase in computer chip memory every 18 months. Affymetrix’s prototype chips held 20,000 oligonucleotide DNA probes. By 1997, this number had increased to 65,000 probes and it has recently introduced a chip with 400,000 probes. Ten such chips could scan the entire human genome for ‘malfunctioning’ genes.

（Source：Keith Firman, Nanonet)