MicrobiologyBytes

Microarray technology has evolved since the 1980s from Southern blotting, an analytical technique in which fragments of DNA are attached to a substrate and then probed with a known gene or DNA fragment to reveal their presence. A DNA microarray (also commonly known as gene chip or gene array) is a collection of microscopic DNA spots, usually representing single genes, regularly arranged on a solid support such as a glass microscope slide and covalently attached via a chemical matrix. Glass has low intrinsic fluorescence, is chemically inert, and the standardized dimensions of microscope slides simplify manufacture and handling of arrays. Tens of thousands of DNA probes, usually chemically synthesized oligonucleotides 20-70 nucleotides in length, can be attached to a slide and the genes they represent can all be analyzed in a single experiment.

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To achieve the very high density of DNA probes in the array, spotting or printing of the slides is carried out by specialized robots:

DNA microarrays can be used in comparative genome analysis, but are most commonly used to detect messenger RNAs (mRNA), and this is referred to as expression analysis or expression profiling (Microarray Analysis: Genome-scale hypothesis scanning. PLoS Biol 2003 1: e15). The usual method is to fluorescently label an RNA sample in an enzymic reaction, e.g. reverse transcription, during which the RNA is converted into complementary DNA (cDNA). Amplification of sequences by PCR is sometimes incorporated into this step:

The cyanine dyes Cy3 and Cy5 which have excitation wavelengths of 635 nm and 532 nm respectively are most frequently used to label the RNA samples. Two-color labeling allows two samples, for example, two different tissues, treated and untreated, or infected and uninfected samples to be hybridized to the same array and their gene expression profiles compared via the difference in the fluorescence of the two samples. This complex mixture of sequences is then allowed to hybridize to the DNA capture probes on the array for up to 24 hours:

Unbound (non-complementary) sequences are washed away and the fluorescence of the individual DNA spots on the array is measured by laser excitation:

Statistical post-processing of the fluorescence data is usually necessary to eliminate artifacts and false results from the data obtained.

Because of the power of this technique, microarrays have become the pre-eminent technology for the investigation of functional genomics – the area of modern biology which focuses on dynamic aspects such as gene transcription, translation, and protein-protein interactions, as opposed to the static aspects of the genome such as DNA sequence or structure. Use of a collection of distinct DNAs in arrays for expression profiling was first described in 1987, when arrayed DNAs were used to identify genes whose expression was modulated by interferon (Identification of interferon-modulated proliferation-related cDNA sequences. PNAS USA 1987 84: 8453-8457). These early gene arrays were made by spotting cDNAs onto filter paper with pins. The first analysis of a complete genome (that of yeast, Saccharomyces cerevisiae) using a microarray was published in 1997 (Yeast microarrays for genome wide parallel genetic and gene expression analysis”. PNAS USA 1997 94: 13057-13062).

Bacterial genes are regulated at the transcriptional level, so understanding the nuances of gene expression is very important in understanding pathogenesis. Using microarrays to monitor bacterial gene expression during infection has both advantages and potential pitfalls (Benefits and pitfalls of using microarrays to monitor bacterial gene expression during infection. Current Opinion in Microbiology 2004 7: 277-282). The technology allows comparison of RNA preparations from bacteria cultivated in vitro with those taken from an infected individual, thus pinpointing the changes which occur during infection. Previously, data on bacterial gene expression during infection had to be obtained from individual reporter gene assays. The array method is particularly valuable for bacteria which replicate intracellularly, such as Mycobacterium tuberculosis, Salmonella, and many more. However, comparing RNA profiles from in vitro cultures and ex vivo cells is not straightforward, nor is quantitative preparation of sub-microgram levels of bacterial RNA from infected animals or plants. Internal controls used to standardize microarray data are of critical importance in this type of experiment.

Plant viruses are a very diverse group which, unlike bacteria, possess no nucleotide sequences in common, e.g. ribosomal RNA genes. Detection of plant viruses is becoming more challenging as globalization of trade and the potential effects of climate change enhance the movement of viruses and their vectors. Methods based on PCR provide the greatest sensitivity, but are limited in parallel detection capability even in “multiplexed” applications. Microarrays provide the greatest capability for parallel yet specific identification, and can be used to detect individual or combinations of viruses and, using current approaches, to do so with a sensitivity comparable to enzyme-linked immunosorbent assay (ELISA) (Microarrays for Rapid Identification of Plant Viruses. Ann. Rev. Phytopathol. 2007 45: 307-328).

Microarray analysis is an enormously powerful technique, but the complexity and cost of the technology involved puts it beyond the reach of many laboratories, and moves microbiology into the Big Science league, alongside space exploration and particle physics.

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This entry was posted on Monday, November 19th, 2007 at 10:16 am and is filed under Bacteria, Biology, Biotechnology, Genetics, Health, Medicine, Microbiology, Podcast, Science, Virology. You can follow any responses to this entry through the RSS 2.0 feed. Both comments and pings are currently closed.