DNA microarray an orderly, low density arrangement of DNA samples, for example cDNA (see COPY DNA), immobilized as spots on a matrix. DNA microarrays can be fabricated by robotic spotting of single-stranded DNA PROBE molecules, on to the surface of a microscope slide, such that DNA of known identity is immobilized at precisely defined locations. A single slide may contain information for thousands of genes.
A typical DNA microarray/chip experiment involves matching known (probe) and unknown (target) nucleic acid samples on the basis of COMPLEMENTARY BASE PAIRING through HYBRIDIZATION, and automating the process of identifying the unknowns.
The target single-stranded nucleic acid sample, DNA or RNA, whose identity/ abundance is being determined, is labelled (see LABEL for example using a fluorescent dye, and incubated with the microarray/chip so that hybridization can occur between probe and target. Any unhybridized, free nucleic acid is washed away and HYBRID molecules detected by scanning and locating the positions of the fluorescent label. Abundance depends on the amount of fluorescence, whilst the position of fluorescence identifies the sample.
[Note: the definitions for the terms ‘probe’ and ‘target’ as used here differ from those used in classical hybridization. Here, the probe is immobilized and unlabelled, whilst the target is free and labelled.]
A problem with the use of microarrays is the potential for intra-strand BASE PAIRING in nucleic acid molecules, which could block probe-target interactions and hybrid formation. The use of PNA probes may circumvent this, since PNA:DNA hybrids can form under conditions that inhibit intra-strand base pairing in single-stranded DNA.
Microarrays/chips allow a large number of hybridization experiments to be carried out simultaneously in a single assay, so that thousands of GENES could be analysed in parallel. The technology can be used for a number of purposes including
- identification of genes;
- determination of the level of GENE/GENOME EXPRESSION in, for example, normal cells or tissues, diseased states, cell cycle analysis, and response to environmental stimuli;
- Detection of MUTATIONS and POLYMORPHISMS. For gene expression studies mRNA or cDNA is prepared and used to hybridize to specific probes. Hybrids identify the expressed genes.
Applications of the technology include drug discovery and development, human and agricultural diagnostics (see DIAGNOSIS), analysis of food and GMOs, SNP identification, and screening for TOXINS (see TOXICOGENOMICS).
DNA microarrays/chips are important tools in functional GENOMICS.
The terms ‘DNA microarray’ and ‘DNA chip’ are often used interchangeably although there is a technical difference. Other terminologies include biochip, gene chip, gene array
DNA binding proteins
are of two general types, histone proteins which are part of the unit structure of chromosomes called nucleosomes and nonhistone proteins which are present in small amounts and include regulatory proteins.
a DNA molecule that is a closed-ring structure, found in mitochondria, prokaryote chromosomes, plasmids, and certain viruses.
closed DNA complexes
the first of two kinetically distinct steps required for RNA polymerase to initiate transcription in which the RNA polymerase holoenzyme binds electrostatically to the promoter DNA.
a DNA molecule which has been inserted into a cloning vector.
a DNA copy of mRNA which contains only regulatory and coding sequences, i.e. introns have been removed. mRNA is copied into double-stranded DNA using reverse transcriptase; the cDNA can then be cloned and amplified and introduced into an expression vector (plasmid or phage) and its protein product produced in either bacterial, yeast, insect or mammalian cells. Called also cDNA.
end labeling DNA
methods for labeling DNA with radioisotopes or other detectable marker molecules at the ends using the terminal transferase 3′-labeling or polynucleotide kinase for 5′-labeling.
that present in a cell as extra chromosomal; exemplified by plasmids of prokaryotic cells. See plasmid
the DNA that has been introduced into a host by cloning.
enzymes involved in the excision-repair mechanisms for DNA.
duplex DNA with each strand from a different origin.
a collection of cloned DNA molecules from a genome.
an enzyme that seals nicks in the DNA helix, joins Okazaki fragments together during DNA replication and is essential in recombinant DNA technology for DNA cloning.
an ordered set of thousands of different oligonucleotides immobilized on a microscope slide or other solid surface used for the detection of cognate nucleotide sequences such as the pattern of gene expression in a particular cell population by hybridization with fluorescently labeled cDNA prepared from total mRNA isolated from the cells.
a sequence present in the variable locations on the chromosome. Called also jumping genes. See also retrotransposon and transposable genetic elements.
open DNA complex
a local opening of about 10 base pairs formed at the transcription initiation site following the electrostatic binding of RNA polymerase holoenzyme to the promoter region.
of Escherichia coli; has three distinct enzymatic activities: (a) a 5′ to 3′ polymerase activity which, under the direction of a template DNA, catalyzes the addition of mononucleotide units, produced from deoxynucleoside 5′-triphosphates, to the 3′-hydroxyl terminus of a primer chain; (b) a 5′ to 3′ exonuclease active only on duplex DNA; (c) a 3′ to 5′ exonuclease primarily active on single-stranded DNA which can selectively remove mismatched terminal nucleotides, thus carrying out a proofreading function. Additionally it catalyzes both the pyrophosphorolysis of DNA, a reaction which is the reverse of polymerization, and pyrophosphate exchange which represents a repetitive sequence of nucleotide addition and pyrophosphorolysis.
a series of enzymatic mechanisms whereby errors or damage to one of the two DNA strands are removed by excision and replaced by correct nucleotides using the undamaged strand as template. The mechanisms include removal of lesions of depurination and DNA glycosylases which recognize altered bases.
repeat DNA, repetitive DNA
includes (a) satellite DNA and so-called (b) interspersed repeated DNA sequences. The latter are interspread throughout the chromosomes in hundreds of thousands of individual copies, each about 300 nucleotides long; they are, unlike satellite DNA, transcribed.
serially repeated DNA sequences of one or a few nucleotides with a repeat length of up to 250 nucleotides that are not transcribed and commonly located in the heterochromatin associated with the centrometric regions of chromosomes.
a mobile DNA element that appears to have no function except to replicate itself. Part of junk DNA.
determining the order of nucleotides in DNA from which amino acid in a polypeptide chain can be predicted.
the fraction of DNA that contains most of the protein-coding genes and reassociates most slowly.
produced when double-stranded DNA is denatured or found naturally in some viruses.
single-copy DNA sequences which do not encode proteins or functional RNA molecules.
the double helix is itself twisted.
a twisted structure formed by circular DNA molecules. See also supercoiled DNA (above).
DNA sequences that occur only once in the haploid genome.
contain a single molecule of DNA that is either double or single stranded. Parvoviruses and circoviruses are single stranded, hepadnaviruses are partially double stranded and all others are double stranded. DNA virus families are: Poxviridae, Asfarviridae,Herpesviridae, Adenoviridae, Papovaviridae, Parvoviridae, Circoviridae, and Hepadnaviridae.
an alternative structural form of DNA which differs from the more commonly occurring B- and related A-form in that the helix is left handed compared with the right hand helixes of B- and A-forms. Z is for zig-zag. The functional significance of Z-DNA is unknown.