DNA MICROCHIP TECHNOLOGY (Part ii)

3) What actually the DNA microchip technology is?

Currently, DNA microchips are referred by several different terms. Some have subtle difference of meaning, but the student can regard the terms,
-DNA chip
-DNA array
-DNA microarray
-Genome chip (as effectively the same).
The first step in manufacturing of the microarray is the determination of the DNA samples used, by the purpose for which the chip is to be used for. These can range from short oligomers, to large PCR products, cDNA molecules or cloned inserts.
Manufacture requires sophisticated robotic equipment that will permit up to one million spots each containing specific DNA molecules to be attaches in an area less than 1.5 cm2.
Two different methods can be used to construct the array.

3.1)DNA molecules can be deposited onto specific sites in the array by a method similar to ink-jet printing. This is used for larger DNA molecules.
3.2)For short oligomers, photolithography, a technique borrowed from the semiconductor industry, is used to synthesize the DNA in situ.

The hybridization step requires the target nucleic acid to be labeled. This can be with a radioactive label, but fluorescent labels are now more common. These reduce handling problems and allow for improved detection methods. Hybridization conditions differ depending on the type of probe attached to the glass. In essence, hybridization conditions are designed to produce a minimum of non-specific hybrid, and unhybridized molecules from the target population are washed away at the end of the reaction.
Because of the high density of DNA spots in the microarray, identification of spots which have undergone hybridization is done by computer. If a fluorescent label is attached to the target molecules the microarray is scanned by fluorescence microscopy. Where the target has hybridized strongly to a probe a bright fluorescent signal is detected. The software then correlates the signals with the specific DNA molecules at each spot in the microarray.

4) The Use of DNA Microchip in various fields:

4.1) In Medical Science:

4.1.1) Cystic Fibrosis, in this disease many different mutations are known within the gene responsible. A chip could contain examples of all mutations known for this gene, thus making screening easier to perform. This methodology is currently available to determine if the p53 gene is mutant in tumor samples. In this case a single chip will detect point mutation in any part of the entire coding sequence of the gene.
4.1.2) Using DNA-microchip technology expression maps in cells of bone marrow or immobilized blood of patients with different types of leukemia at different stages will be obtained and compared with results obtained from cells without effect of cancer. The differences found by comparing expression maps of cells with various level of progression and differentiation will help us to identify the primary, yet known, but also new genes whose false function contributes to the disease development. Also it should be clarified how the changes of activity of these genes reflect the activity of other known or unknown genes and what is the location of these genes on corresponding chromosomes. For epigenetic changes FISH and high-resolution cytometry will be used. Chromosomes with modulated level of expression will be interrogated structural-epigenetic-study. Changes in structure of chromatin in the cells of patients with leukemia in the various stages of diseases in contrast to non-cancer cells will be evaluated

4.2)In Astronomical Science: During the space voyage it is important to have the Microbial monitoring of spacecraft and space stations to maintaining system integrity and crew member health. Ribosomal RNA-based methods of microbial detection are particularly effective. A new DNA microchip technology now provides advantages over more traditional hybridization formats. This method utilizes a micro-array of gel elements containing hundreds of individual DNA-probes bound to a glass slide. The probes are complementary to the 16S rRNA of a selected bacterial species, genus, or higher taxonomic grouping. This format provides for massively parallel hybridization and the simultaneous identification of many microbial population types.

4.3)In Microbiology: Scientists are developing a DNA microchip technology to monitor microbial contaminants of water systems.

4.4)In Genetics: Most of the early experiments using DNA microarrays were designed to examine patterns of gene expression.
4.4.1) Genotype analysis: DNA microchip technology can easily be adapted to the detection of Single Nucleotide Polymorphisms (SNPs). These are mutation in which one base in a sequence of DNA has been deleted replaced with another base. Polymorphisms of this nature are becoming increasingly important in mapping genes. DNA microchips, with the very high numbers of genes that can be monitored on the same computerized chip provide such a system. For the detection of SNPs short probes are used. These consist of sequences about 25 bases (oligomers) that are usually synthesized in situ. The nucleotide for which a polymorphism is being investigated is situated near the center for the oligomer. A series of four oligomers are syntheize on adjacent spots of the array. In addition to being used to score genotypes, and thus create genetic maps, DNA microarray technology can also be used to identify new SNPs in a population.
4.4.2) Genetic Testing: The use of genetic tests to confirm diagnoses of inherited diseases, or to identify carriers or individuals with late onset diseases, is now of considerable significance in medicine. A variety of techniques built upon sequence analysis have been developed to this end. DNA microarray technology may also be applied in this area. In the same way as in it detects SNPs the system can detect mutations in genes of clinical significance. This is of specific importance where an entire gene needs to be screened for mutations, rather than only a small number of sites where mutation is known to occur. An example of this is cystic fibrosis. In this disease many different mutations are known within the gene responsible. A chip could contain examples of all mutations known for this gene, thus making screening easier to perform. This methodology is currently available to determine if the p53 gene is mutant in tumor samples. In this case a single chip will detect point mutation in any part of the entire coding sequence of the gene.