The use of microarrays or DNA chips allow large-scale analysis and quantification of genes and gene expression. A microarray consists of an ordered arrangement of potentially hundreds of thousands of DNA sequences, such as oligonucleotides or cDNAs deposited onto a solid surface. The solid support may be either glass or silicon, and currently the arrays are synthesised on or off the chip. They require complex fabrication methods similar to those used in producing computer microchips. Most commercial production employs robotic ultrafine microarray deposition instruments that dispense volumes in the picolitre range. Alternatively, on-chip fabrication, as used by Affymetrix, builds up layers of nucleotides using a process borrowed from the computer industry termed photolithography. Here, wafer-thin masks with holes allow photoactivation of specific dNTPs, which are linked together at specific regions on the chip. The whole process allows layers of oligonucleotides to be built up with each nucleotide at each position being defined by computer control.

The arrays themselves may represent a variety of nucleic acid material. This may be mRNA produced in a particular cell type, termed cDNA expression arrays , or may alternatively represent coding and regulatory regions of a particular gene or group of genes. A number of arrays are now available that may be used for determination of mutations in DNA, mRNA transcript levels or other polymorphisms such as SNPs. Sample DNA is placed on the array, and any non-hybridised DNA washed off. The array is then analysed and scanned for patterns of hybridisation by detection of fluorescence signals. Any mutations or genetic polymorphisms in relevant genes may be rapidly analysed by computer-controlled interpretation of the resulting hybridisation pattern and mutation, transcript level or polymorphism defined. Indeed, the collation and manipulation of data from microarrays presents as big a problem as fabricating the chips in the fi rst place. The potential of microarrays appears to be limitless and a number of arrays have been developed for the detection of various genetic mutations, including the cystic fibrosis CFTR gene (cystic fibrosis transmembrane regulator) and the breast cancer gene BRCA 1, and for the study of the human immunodeficiency virus ( HIV).

One current application of microarray technology is the generation of a catalogue of SNPs across the human genome. Estimates indicate that there are approximately 85 million SNPs and, importantly, some may point to the development of certain dis eases. For example, a SNP in the F5 gene is implicated in a thrombophilia, factor V Leiden, a genetic blood clotting disorder. SNP analysis is therefore clearly a candidate for microarray analysis and developments such as the Affymetrix Genome-Wide SNP array enable the simultaneous analysis of nearly 1 million SNPs on one gene chip. In order to simplify the problem of the vast numbers of SNPs that need to be analysed, the HapMap project currently analyses SNPs that are inherited as a block, and in theory as few as 500 000 SNPs will be required to genotype an individual.

An extension of microarray technology may also be used to analyse tissue sections. This process, termed tissue microarrays (TMAs), uses tissue cores or biopsies from conventional paraffin-embedded tissues. Thousands of tissue cores are sliced and placed on a solid support such as glass where they may all be subjected to the same immunohistochemical staining process or analysis with gene probes using in situ hybridisation. As with DNA microarrays, many samples may be analysed simultaneously, less tissue is required and greater standardisation is possible.

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مواضيع ذات صلة

Analysing Genetic Mutations and Polymorphisms

The Largest Subunit in RNA Polymerase II Has an Essential Carboxy-Terminal Repeat

Three Eukaryotic RNA Polymerases Catalyze Formation of Different RNAs

Regulatory Elements in Eukaryotic DNA Are Found Both Close to and Many Kilobases Away from Transcription Start Sites

Control of Gene Expression in Bacteria

Nucleic Acid Electrophoresis

Analysing Genes and Gene Expression

Screening Gene Libraries

Hybridisation and Gene Probes

Cloning Vectors: Vectors Used in Eukaryotes

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes

Centromere Sequences Vary Greatly in Length and Complexity