Mark Schena

Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray

A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.

Identification of the SAAT Gene Involved in Strawberry Flavor Biogenesis by Use of DNA Microarrays

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

Overview of DNA chip technology

DNA chip technology utilizes microscopic arrays (microarrays) of molecules immobilized on solid surfaces for biochemical analysis. Microarrays can be used for expression analysis, polymorphism detection, DNA resequencing, and genotyping on a genomic scale. Advanced arraying technologies such as photolithograpy, micro-spotting and ink jetting, coupled with sophisticated fluorescence detection systems and bioinformatics, permit molecular data gathering at an unprecedented rate. Microarray-based characterization of plant genomes has the potential to revolutionize plant breeding and agricultural biotechnology. This review provides an overview of DNA chip technology, focusing on manufacturing approaches and biological applications.

Fluorescence-based expression monitoring using microarrays.

Publisher Summary Although microarrays show great promise as tools for genotyping, mapping, and resequencing, an equally important application for the microarray is measuring transcript abundance. Microarrays have been used to simultaneously measure the mRNA expression levels for every gene in Saccharomyces cerevisiae under several different growth conditions, to characterize the differences between normal and metastatic tissues and as a vehicle for gene discovery. This chapter describes the two basic types of microarrays, protocols for fluorescently labeling messenger RNA from eukaryotic cells for hybridization to microarrays, and considerations involved in experimental design and data analysis. Many variations on the protocols are feasible: poly(A) purification steps might be eliminated, different fluors may work as well or better, and polymerase chain reaction (PCR) amplification of cDNA may be possible. A potential problem for all microarray experiments is cross-hybridization. Because microarrays generally have longer probes than do oligonucleotide arrays, some have argued that their specificity is greater and that the potential for cross-hybridization is lower.

Parallel analysis with biological chips

Publisher Summary This chapter discusses how the authors reached the situation where they needed a biological chip. The chapter describes how they devised their first chip-based assays for biological analysis. The basic concept of a biological chip assay is explained here. Biological extracts are deciphered by reacting these mixtures with an ordered array of biological molecules. The extent of binding of labeled products to their cognate array elements provides a quantitative readout of important cellular processes such as gene expression. Parallelism allows precise comparisons to be made between all the genes or gene products represented in the array. Pseudocolor representations simplify data analysis. Also, colorful illustrations have been used in this chapter to help readers visualize a biological chip assay. Moreover, formal methodological guidelines are provided to expedite biological chip experiments. Finally, the chapter outlines various applications including gene expression monitoring, DNA resequencing, point mutation analysis, and genotyping applications. Biological chips have immediate applications in both academic and commercial settings. These allow for accelerated data acquisition for drug discovery, disease diagnosis and prognosis, and basic research. And, biological chips used in massive, parallel assays will assist in accelerating the information age of biology.

Strawberry on chips: gene expression analysis during strawberry development using cDNA microarrays

Strawberry on chips: gene expression analysis during strawberry development using cDNA microarrays