Rice Genetics IV - IRRI books - International Rice Research Institute

Microarray technologyThe rapid accumulation of genomic sequences has been paralleled by the growth incollections of ESTs, each of which contains the partial sequence of an expressed gene(Adams et al 1993, Marra et al 1998). The public dbEST includes approximately900,000 plant EST sequences among the more than 6.6 million ESTs deposited intotal. For these sequences to be informative, the genes that they identify must beassigned biological functions. This will ultimately be a biochemical or structural function,but many sequences are now annotated in descriptive terms that must not bemistaken for a functional identity. Realistically, we know the precise function of maybea quarter of all genes and we know some characteristics for about another quarter. Atleast one-half of all genes, however, remain functionally unknown. Expression profilingusing DNA microarrays provides a powerful tool for correlating some aspectsof gene functions, such as tissue-, developmental-, or external manipulation-specificexpression, with sequences. DNA microarrays are also useful for identifying differentiallyexpressed genes in organisms for which genomic DNA sequences are limited.The use of microarrays for expression profiling is based on two fundamental principles.The first is that, for many genes, a predominant factor underlying changes inexpression is an alteration in the abundance of the cognate mRNA. It is clear thatpost-transcriptional factors also affect gene expression but the analysis of these factorsis not amenable through microarray analysis. The second principle is that onlyDNA strands possessing complementary sequences can hybridize to each other toform a stable, double-stranded molecule. Microarrays exploit this property throughthe immobilization of single-strand copies of a gene as individual array elements onsurfaces such as glass slides coated with polylysine. These elements are termed the“probe” (Phimister 1999). After incubation with a mixture of (radioactively orfluorescently) labeled DNA molecules (termed “target”), representing a proportionalrepresentation of all genes present in a given (tissue) sample, the labeled moleculesthat represent the same gene as the immobilized DNA elements can form heteroduplexes.By measuring the amount of label that is bound to each array element at theend of the hybridization reaction, the relative transcript abundance of each gene canbe determined. In this way, RNA abundance levels for thousands of genes can bemeasured in a single experiment. Patterns of gene expression can be correlated withparticular tissues or experimental conditions by comparing abundance levels fromdifferent experiments.Two types of microarrays, which differ primarily in the length of the probes thatmake up the array elements and in the production of these elements, are being usedfor expression profiling. For DNA microarrays, printed on membranes or glass, theelements are large gene fragments (approximately 400 to 2,000 bp or more), typicallyproduced through polymerase chain reaction (PCR) amplification. In contrast, oligonucleotide-basedarrays (termed “chips”) (Lipshutz et al 1999) contain elements madeup of short synthetic DNA molecules. These are synthesized such that a set of 10 ormore oligonucleotide fragments report the presence of one gene and they includecontrols, which have one base mismatch for each of the selected fragments. The pro-Isolation of candidate genes for tolerance of abiotic stresses 353