A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization.

Abstract

Detecting and determining the relative abundance of diverse individual sequences in complex DNA samples is a recurring experimental challenge in analyzing genomes. We describe a general experimental approach to this problem, using microscopic arrays of DNA fragments on glass substrates for differential hybridization analysis of fluorescently labeled DNA samples. To test the system, 864 physically mapped X clones of yeast genomic DNA, together representing >75% of the yeast genome, were arranged into 1.8-cm x 1.8-cm arrays, each containing a total of 1744 elements. The microarrays were characterized by simultaneous hybridization of two different sets of isolated yeast chromosomes labeled with two different fluorophores. A laser fluorescent scanner was used to detect the hybridization signals from the two fluorophores. The results demonstrate the utility of DNA microarrays in the analysis of complex DNA samples. This system should find numerous applications in genome-wide genetic mapping, physical mapping, and gene expression studies. As a general approach to this problem, we have developed a system for making microarrays of DNA samples on glass substrates, probing them by hybridization with complex fluorescentlabeled probes, and using a laser-scanning microscope to detect the fluorescent signals representing hybridization. Fluorescent labeling allows for simultaneous hybridization and separate detection of the hybridization signal from two or more probes. This in turn allows very accurate and reliable measurement of the relative abundance of specific sequences in two complex samples. with a mixed probe consisting of lissaminelabeled DNA from the 6 largest yeast chromosomes together with fluorescein-labeled DNA from the 10 smallest yeast chromosomes. A red color indicates that yeast sequences present in the lissamine-labeled hybridization probe hybridized to an array element. A yellow-green color indicates that yeast sequences present in the fluorescein-labeled hybridization probe hybridized to an array element. An orange color indicates cross-hybridization of both chromosome pools to an array element (e.g., dispersed repetitive elements, such as Tyl elements). RESULTS Array Hybridization PatternEach clone was spotted twice, resulting in duplicate hybridization patterns in adjacent quadrants of the array. Control DNA spots, which were randomly amplified in the same manner as the X clone array elements, are located in the bottom corner of each quadrant. "A" points to a pair of spots containing total yeast genomic DNA. These spots appear orange because both chromosome pools hybridized to yeast genomic DNA. The negative controls are as follows: "B" points to a pair of spots of wild-type X DNA, "C" points to a pair of human genomic DNA spots, and "D" points to a pair of ~X174 DNA spots. The lack of a hybridization signal at these three negative control spots indicates that the hybridization was specific for yeast sequences.

Profiling of gender-regulated gene transcripts in the filarial nematode Brugia malayi by cDNA oligonucleotide array analysis

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Proteasome inhibitor induced gene expression profiles reveal overexpression of transcriptional regulators ATF3, GADD153 and MAD1

“…Fluorescent probes for hybridization were derived from two sources of mRNA ± MCF7 cells treated with DMSO or 10 mM Lactacystin for 21 h ± which were labeled by reverse transcription with two di erent¯uorophores to provide a direct and internally controlled comparison of the mRNA expression level of every gene present on the array. In principle, the technology has been described (Schena et al, 1995;Shalon et al, 1996). The average standard deviation of the relative¯uorescent signal of individual data points between independent, but identical experiments was below 20%.…”

Expression scanning of an array of growth control genes in human tumor cell lines

“…The most productive implementation uses hybridization of complex probes prepared from cell or tissue RNA to`macroarrays' of cDNA inserts on Nylon membranes (Bernard et al, 1996;Tsou et al, 1998), miniaturized`microarrays' constructed on glass slides (Schena et al, 1995;DeRisi et al, 1996;Shalon et al, 1996) or on Nylon membranes (Chen et al, 1998), or`DNA chips' carrying oligonucleotides (Lockhart et al, 1996;Wodicka et al, 1997). In all cases, hybridization intensity can be used to derive expression data for each gene represented on the array.…”