Arrays are a relatively recent phenomenon in the field of molecular pathology.83 The term "array" is jargon for an orderly collection of molecules on solid supports ranging from nylon membranes to printed circuit board to glass slides to silicon surfaces. There are macroscopic arrays, for example, reverse line blots on nylon membranes, and microarrays, for example, DNA chips. Synonyms for microarrays include gene chip, DNA chip, genome chip, biochip, gene array, DNA array, and DNA microarray.
Though most do not think of line probe assays as belonging in a section describing DNA chips, they are indeed arrays. Line probe assays are being used to determine mutations in the HIV genome, to type HCV, and to detect CFTR mutations for cystic fibrosis.84 The method is based on classic nucleic acid hybridization using nylon or nitrocellulose strips as a solid support matrix for hybridization and detection. Generally, reverse hybridization is employed. Oligonucleotide probes specific for mutations or polymorphisms of interest are bound to the membrane strip in a parallel line format. Patient specimen DNA is amplified with biotinylated primers, thereby labeling the amplicon, which is hybridized to probes on the membrane. After hybridization, alkaline phosphatase-labeled streptavidin is added and binds to the biotin of the bound amplicons. Detection is colorimetric following incubation with a specific chromogen, generating a purple-
brown precipitate. The resulting colored lines are read as positive results, hence the name of the assay.
DNA chips, such as those produced by Affymetrix, Nanogen, Clinical Micro Sensors, and other companies, may be thought of as miniaturized ASOH platforms with multiple oligonucleotide probe assays. In one model, the oligonucleotides are synthesized directly onto a solid support, for example, glass or silicon, that is, the "chip." Some companies' chips can hold thousands of unique sequences. The sample DNA is labeled with fluorescent dye, denatured, and hybridized with the oligonucleotides on the chip. The chip is then scanned and the fluorescence patterns are measured with the aid of computer analysis to determine the presence of mutant or wild-type sequences. Gene expression chips use probes that bind to many different cDNA species (up to 33,000 in one array at the time of this writing) to quantitate the level of each cDNA isolated in a sample. Gene expression microarrays with large densities are appropriate for analysis of differential patterns of gene expression between normal and diseased tissues.
Clinical molecular laboratory diagnostic applications for DNA chips currently are limited to those with clinically appropriate density, in the range of 10 to 200. Utility for these platforms is found in SNP and mutation detection85 as well as pathogen identification. Chips with densities in the thousands have limited, if any, use in clinical laboratories at this time. So much data are generated that more analysis needs to be done to winnow down the numbers to a relevant few genes or transcripts that can be used in clinical assays. This work is progressing well. Furthermore, the informatics software currently being used to analyze these data requires simplification and modification so that useful algorithms for data analysis can be employed for disease diagnosis or prognosis. While these are certainly considerable challenges, it should be noted that DNA array chip technology benefits enormously from robotic manufacturing techniques and rapidly advancing computer software. This has greatly decreased the costs per array feature of DNA chips, giving DNA microarrays enormous potential in the clinical molecular laboratory of the near future.
Examples of Applications of Arrays
1. Line probe assay for HCV genotyping
2. DNA chips for detection of polymorphisms in cyto-
chrome P450 genes for pharmacogenetics
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