D.N.A. MICROCHIP TECHNOLOGY
The DNA microchip is a new tool used to identify mutations in genes. The chip, which consists of a small glass plate encased in plastic, is manufactured somewhat like a computer microchip. On the surface, each chip contains thousands of short, synthetic, single-stranded DNA sequences, which together add up to the normal gene in question. 1) Introduction Nucleic acid hybridization, where two complementary single-stranded nucleic acid molecules form a stable hybrid has been the basis of the advances made in molecular genetics. The two major techniques involved are Southern and Northern blotting. The former identifies specific DNA sequences in a sample, and the latter the presence of specific RNA molecules. Both of these procedures have been extremely useful, but they have technical deficiencies. The problems are largely associated with the length of time taken to carry out the process, and the fact that only one probe (gene) can be used at a time. This was not so important when relatively few genes had been characterized, but with the amount of data that has been made available through the genome projects more sophisticated methods of analysis are now required. Northern and Southern blotting procedures have limited usefulness because of the length of time they require and the fact that they only deal with one probe at a time. DNA microchips allow the rapid analysis of thousands of genes simultaneously. These chips are small wafers to which large numbers of different DNA molecules have been attached in organized arrays. DNA or RNA taken from tissues is hybridized to the chips. The attached DNA molecules are referred to as probes and the nucleic acids that are hybridized to them, as targets . DNA is either deposited on the chips using computer-controlled jets or is synthesized at a specific spot in the array by photolithography. Target molecules are usually fluorescently labeled. At the end of the procedure probes that have hybridized to target molecules are identified by fluorescence microscopy. Here the probes represent specific genes. The targets are derived from mRNA isolated from tissues. This mRNA is usually fluorescently labeled by copying it to cDNA using reverse transcriptase. All the genes that were expressed in the tissue will appear as fluorescing spots in the chip. This process is normally used to compare cells under two different sets of conditions. It has shown that over 1000 genes in yeast are expressed differently when the when cell sporulates . Similarly comparisons of patterns of gene expression can be made between normal and tumor cells. Competitive hybridization utilizes targets that fluoresce with different colors to determine accurately- levels of gene expression. DNA microchip technology is an important method for recognizing single nucleotide polymorphisms (SNPs) , where alleles differ by only one nucleotide. The probes are synthetically produced short oligomers that contain the nucleotide of interest. Five copies of the coding strand will be synthesized at adjacent spots in the array. Four of these will contain different substitutions of the base in question and one will omit the base. A second set of spots will be occupied by the same permutations of the non-coding strand. These can be used to genotype DNA taken from individuals to determine if they are homozygous or heterozygous for any SNPs at that locus. A DNA chip can be constructed to genotype thousands of different loci simultaneously. DNA microchip technology can identify mutations in genes of medical importance. This is particularly important where mutations at many different sites in a gene are known to cause a disease. Cystic fibrosis is a good example of this. It is also possible to use this technique to scan tumor suppressor genes such as p:53. 2) Background behind the development: The development of DNA microchip technology has allowed the analyses of DNA and RNA to be carried out at high speed and with great accuracy. Several thousand genes may be analyzed in a single experiment. This has had its main effects in analysis of patterns of gene expression, identification of genotypes and in genetic screening. A DNA microchip is essentially a matrix, usually glass, onto which very large numbers of DNA samples have been attached in an organized array. These organized arrays of thousands of different DNA molecules are used in experiments where they are hybridized to DNA or RNA samples, and those DNA molecules that form hybrids are detected. This information is then used to gain biological information about the nucleic acid molecules in the sample. There is one important difference between DNA microarray technology and conventional Southern and Northern blotting. In microarrays the DNA samples that make up the array are known as probes and the nucleic acids added in solution as targets. In conventional blots the probe is in tthe solution.