Mutation analysis of genes has accelerated our understanding of disease-related mechanisms, which has had an impact in terms of both basic knowledge and clinical practice. However, each gene has different mutation characteristics and a different size. Most of the APC, BRCA1 and BRCA2 mutations found in cancers are protein truncating nonsense or frameshift mutations. However, missense mutations dominate in hot-spot areas for the RET proto-oncogene responsible for MEN2 syndromes (8). Several mutation detection techniques have been developed, such as PCR-SSCP (Polymerase Chain Reaction-Single Strand Conformational Polymorphism), PTT (Protein Truncation Test), PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), and sequencing. These conventional methods are widely used because they are easily performed and the experimental conditions are well known. The predominant RET mutations are missense mutations and are restricted to 10 codons (codons 609, 611, 618, 620, 630, 631, 634, 768, 804 and 918) in MEN2 syndromes. Missense mutations at codons 609, 611, 618, 620 and 634 have been identified in 98% of MEN2A families and in 85% of FMTC families. More than 95% of MEN2B patients also have a predominant mutation at codon 918 (Met→Thr). The RET oligonucleotide microarray can detect RET missense mutations at these 10 codons (8). Sixty-five oligonucleotides were designed for the 65 mutation types at 10 codons, and 12 oligonucleotides were designed for the wild-types and positive controls. The RET oligonucleotide microarray can function as a fast and reliable genetic diagnostic device, which simplifies the process of detecting RET mutations (Fig. 1).

Not only does β-catenin function as a downstream transcriptional activator in the Wnt signaling pathway, it is also a submembrane component of the cadherin-mediated cell-cell adhesion system (11,12). β-catenin mutations have been identified in a variety of human malignancies, with most of these being missense mutations restricted to hot-spot areas in exon 3. In many human cancers, including endometrial, gastric, ovarian, hepatoblastomas and colorectal cancers, the majority of β-catenin mutations have been reported at specific GSK-3 phosphorylation sites, i.e., Ser-33, Ser-37, Thr-41, Ser-45 and other residues (Asp-32 and Gly-34) (13). More than 70 β-catenin mutations have been reported in colorectal cancers, and about 90% of β-catenin mutations are found in 11 codons (codons 29, 31, 32, 33, 34, 35, 37, 38, 41, 45 and 48) as missense mutations or in-frame deletions. We have developed an oligonucleotide microarray for detecting β-catenin mutations at these 11 codons (14). This microarray can detect a total of 110 types of β-catenin mutation, including the previously reported 60 mutations. All oligonucleotides on that array were 21 bp long and the mismatch sequence was located in the middle of the oligonucleotide. Oligonucleotides were modified with 5'-amino residues for chemical binding to the slides. Twelve-carbon spacers were used to increase the efficiency of the hybridization and to make it easier for target samples (labeled with fluorescent dye) to access the binding site. One hundred and ten oligonucleotides were designed for codons 29, 31, 32, 33, 34, 35, 37, 38, 41, 45 and 48, and eleven oligonucleotides were designed to detect in-frame deletions in the eleven codons. In total, the 121 designed oligonucleotides covered all substitutions and in-frame deletions in the above eleven codons of exon 3.

Activating mutations of the K-ras gene occur in approximately 20~50% of colorectal cancers, with ~85% of the mutations restricted to codons 12 and 13 (15). Studies of associations between K-ras mutations and specific clinical features generally require analysis of large numbers of samples (16). Thus, researchers need a high-throughput technique for assessing K-ras mutations. Oligonucleotide microarrays may provide a valid option, as they allow scientists to accurately and rapidly process large numbers of samples. We developed a new method of K-ras oligonucleotide microarray analysis called Competitive DNA Hybridization (CDH). CDH is a novel, efficient, high capacity hybridization technique involving the mixing of various fluorescent-labeled samples to compete with each other in the hybridization reaction. For this work, we used dNTPs labeled with Cy5-dCTP, Cy3-dCTP and Alexa™ 594-dUTP, which have distinct spectra. Accordingly, our CDH results showed improved microarray imaging due to less non-specific hybridization.

An Affymetrix GeneChip™ is produced by synthesizing tens of thousands of short oligonucleotides in situ on glass wafers, one nucleotide at a time, using a modification of semiconductor photolithography (17). There are several differences between GeneChips and homemade cDNA microarrays or other oligonucleotide microarrays. In the GeneChip system, both gene expression arrays and variation detection arrays are available. About 4-5 variation detection arrays (p53, HIV, SNP, CYP450, BRCA1) have been reported as being used, and p53 arrays are used for p53 mutation detection by a wide variety of cancer research groups (18). The Affymetrix P450 GeneChip is used for pharmacogenetic screening. This GeneChip is an efficient and reliable tool for testing CYP2D6 gene variation based on five alleles (19). Developing oligonucleotide microarrays like the GeneChip arrays requires very high level synthesis and photolithography technologies.

Early use of photolithography techniques allowed the production of a GeneChip containing more than 65,000 different oligonucleotides in an area of 1.6 cm² (20). Although originally developed for mutation detection, the same technology was adapted to measure expression levels of cytokine genes in murine T cells (21). In general, 11~16 probes are selected among all possible 25-mers to represent each transcript. In addition to choosing the probes based on their predicted hybridization properties, candidate sequences are filtered for specificity. A core element of the GeneChip design, the 'Perfect Match/Mismatch' probe strategy, is also universally applied to the production of GeneChip arrays. For each probe designed to be perfectly complementary to a target sequence, a partner probe is generated that is identical except for a single base mismatch in its center. These probe pairs, called the Perfect Match probe (PM) and the Mismatch probe (MM), allow the quantitation and subtraction of signals caused by non-specific cross-hybridization. The difference in hybridization signals between the partners, as well as their intensity ratios, serve as indicators of specific target abundance (website at http://www.affymetrix.com/technology/design/index.affx). In addition to allowing hybridization to the most specific regions of nucleotides, the use of short oligonucleotides also allows the representation of multiple regions of a single gene in multiple spots, thus reducing the chance of false positives (22). The main advantage of the GeneChip is its ability to measure the absolute expression of genes in cells or tissues (17). GeneChip technology also eliminates handling of bacterial libraries, amplification of sequences and the risk of cross contamination (22). Oligonucleotide microarrays like the GeneChip allow for the differential detection of gene family members or alternative transcripts that can not be distinguished using cDNA microarrays (2). The entire human genome can now be analyzed on a single array. The GeneChip Human Genome U133 Plus 2.0 array contains 1.3 million distinct oligonucleotides and was used to analyze the expression levels of over 47,000 transcripts as well as variants, including over 30,000 well-characterized human genes (23). In the sample preparation, one or two amplification rounds are used to generate cRNA after reverse transcription. This procedure can be carried out with significantly less starting material than is required for other methods, and the protocols use as little as 50 ng of total RNA, allowing analysis of samples that are small or otherwise difficult to obtain (22). Biotinylated nucleotides are used and the hybridized microarrays are stained with fluorescence-labeled streptavidin (22).

Other commercially available oligonucleotide microarrays are prepared using inkjet technology (Rossetta/Agilent) (24). This system was first described as a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ which are ink-jet printed using standard phosphoramidite chemistry methods. Hughes et al. reported that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several sample amplification and labeling techniques (24). In the sample preparation, different fluorescent-labeled nucleotides (Cy3/Cy5) can be incorporated into the cRNAs in Rosetta arrays, similar to cDNA microarrays.