Box 5.1DNA microarrays and chips

High-density arrays of DNA molecules for parallel hybridization analyses.

DNA microarrays and chips are designed to allow many hybridization experiments to be performed in parallel. Their main applications have been in the screening of polymorphisms such as SNPs (Section 5.2.2) and comparing the RNA populations of different cells (Section 7.3.1). They also have potential in novel DNA sequencing methodologies (Section 6.1.2).

Although the terminology is inexact, microarrays and chips are, strictly speaking, two distinct types of matrix (Gerhold et al., 1999). In both architectures, a large number of DNA probes, each one with a different sequence, are immobilized at defined positions on a solid surface. The probes can be synthetic oligonucleotides or other short DNA molecules such as cDNAs. In the earliest technology, the oligonucleotides or cDNAs were spotted onto a glass microscope slide or a piece of nylon membrane to form a microarray. With this approach, only a relatively low density can be achieved - typically 6400 spots as an 80 × 80 array in an area of 18 mm × 18 mm, which is sufficient for examining RNA populations but less applicable to the high throughput analyses needed to type SNPs.

To prepare really high density arrays, oligonucleotides are synthesized in situ on the surface of a wafer of glass or silicon, resulting in a DNA chip. The normal method for oligonucleotide synthesis involves adding nucleotides one by one to the growing end of an oligonucleotide, the sequence determined by the order in which the dNTP substrates are added to the reaction mixture. If used for synthesis on a chip, this method would result in every oligonucleotide having the same sequence. Instead, modified dNTP substrates are used, ones that have to be light-activated before they will attach to the end of a growing oligonucleotide. The dNTPs are added one after another to the chip surface, photolithography being used to direct pulses of light onto individual positions in the array and hence to determine which of the growing oligonucleotides will be extended by addition of the particular dNTP added at each step. A density of up to 250 000 oligonucleotides per cm2 is possible, so if used for SNP screening, 125 000 polymorphisms can be typed in a single experiment, presuming there are oligonucleotides for both alleles of each SNP.

Chips and microarrays are not complicated to use (see figure below). The array is incubated with labeled target DNA to allow hybridization to take place. Which oligonucleotides have hybridized to the target DNA is determined by scanning the surface of the array and recording the positions at which the signal emitted by the label is detectable. Radioactive labels can be used with a low-density microarray, signals being detected electronically by phosphorimaging. This does not provide adequate resolution for a high-density chip, so with these it is necessary to use a fluorescent label. The fluorescent signal is detected by laser scanning or, more routinely, by fluorescent confocal microscopy.

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From: Chapter 5, Mapping Genomes

Cover of Genomes
Genomes. 2nd edition.
Brown TA.
Oxford: Wiley-Liss; 2002.
Copyright © 2002, Garland Science.

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