DNA MICROARRAY

DNA microarray is simply a high-throughput molecular biology technique that is used to determine how genes work at any given point in time in a cell i.e. how genes are turned off or on in a cell. Similar to the traditional southern blotting technique (which is mainly used to detect the presence of a piece of DNA molecule in a sample), DNA microarray allows scientists to array several thousands of DNA on a microarray or microscopic slide at a very short time and in a precise manner. The technology of DNA microarray has become an indispensable research tool within the life sciences; and this high-throughput technique is also applied in medicine, pharmaceutical companies and even in biotechnology to enhance productivity and understanding of the genetic bases of living organisms inclusive of microbes. With DNA microarray, it is possible for molecular biologists to monitor the expression of thousands of the genes of an organism in real-time i.e. at a simultaneous fashion. DNA microarray is a gene-expression profiling-based technique that is computer-enabled and is generally used to identify multiple and similar genes that are usually co-expressed in a cell at a short span of time; and DNA microarray makes use of high-throughput technology such as the FD10 Next-generation DNA Microarray System which makes it possible to analyze several thousands of genes at a short time (Figure 1).

A microarray which can also be called a gene chip is usually a solid slide that is immobilized with DNA oligomers (particularly complementary DNA, cDNA molecules). The immobilized cDNA molecules or DNA oligomers symbolizes the complete genome of the samples or organism whose cells is to be analyzed using the DNA microarray technique. Microarrays are mini-computer chips that are used by molecular biologists to analyze and/or determine the presence of a particular gene sequence in a sample in a very faster approach (Figure 2). The expression of many genes in a single reaction mixture could now be quickly undertaken and in an efficient way as against the period when it was difficult to analyze several thousands of genes at the same time. With DNA microarray several aberrations in the physiological make up of an organism especially as it relates to their genetic makeup could be deciphered; and such knowledge could be used to compare normal and abnormal genes as panacea to unraveling and solving some of the mysteries surrounding some molecular and non-infectious or mutational diseases of man (e.g. cancer). DNA microarray is a computer-enabled experimentation. Because thousands of unique spots are normally arrayed in orderly rows and columns on a DNA microarray slide (which is generally a solid surface immobilized with cDNA molecules), the exact location and sequence of each of the unique spot (which is indicative of a particular gene) is recorded in a computer database. In DNA microarray, the cDNA derived from the mRNA of known genes is immobilized onto a microarray slide. The sample to be analyzed has genes from both the normal tissue/cell as well as the diseased tissues. Since each of the spots on the scanned imagery of the microarray represents a particular gene, spots with more intensity are obtained for diseased tissue gene if the gene is over expressed in the diseased condition. This expression pattern is then compared to the expression pattern of a gene responsible for a disease; and in this way DNA microarray could be employed to detect or diagnose a particular disease condition in a human host. With microarray technology, molecular biology scientists can comfortably monitor and study how gene is expressed in both healthy cells and diseased cells.

Figure 1: The FD10 Next-generation DNA Microarray System for DNA microarray experimentation.

Figure 2: Illustration of a DNA microarray chip or gene chip used for gene expression assays. The microarrays resemble a microscope and they may even be smaller than the microscope slide. DNA microarrays are usually spatially arranged in a two dimensional (2-D) network fashion or grid that is arranged in arrow and column pattern. Aside the Affymetrix – which is a short oligonucleotide array; other gene expression assays also exist and they include: Fibre Optic Arrays (Illumina), Long Oligonucleotide Arrays (Agilent), and Serial Analysis of Gene Expression (SAGE).

Figure 3: Illustration of the protocol involved in performing DNA microarray technique. This is a two-channel or dual-colour microarray experiment in which normal cells and abnormal cells are simultaneously analyzed and compared. In two-channel DNA microarray experimentation, diseased cells or tissues are compared with healthy tissues with a view to unravel the genetic basis or cause of a particular disease. Less active genes produce fewer mRNA molecules while active genes produce many molecules of mRNA molecules. With DNA microarray, molecular biologists can understand how different genes work at different times. Absence of fluorescence or spots on the microarray shows that none of the mRNA molecules was hybridized to the cDNA; and such genes are inferred inactive.

Briefly, DNA microarray involves the hybridization of a messenger ribonucleic acid (mRNA) molecule to its precursor deoxyribonucleic acid (DNA) template molecule in a microarray via the process of immobilization (Figure 3). Immobilization is a biotechnological approach that is used to immobilize or attach macromolecules inclusive of proteins, enzymes and DNA or genes to solid surfaces or structures. In immobilization technique, these macromolecules are bounded (i.e. attached) to a solid carrier or support that enables them to react effectively with their other reactants. The originator DNA molecule in this case is usually a complementary DNA (cDNA) probe which is generated from the mRNA molecule via the activities of reverse transcriptase (RT) enzyme. Reverse transcriptase is an RNA-dependent DNA polymerase enzyme (i.e. an enzyme that is RNA-directed) that synthesizes a DNA molecule from a messenger RNA (mRNA) molecule. The mRNA molecule serves as the template molecule for the biosynthesis of the DNA molecule.

According to the central dogma of molecular biology as aforementioned in this textbook, DNA is transcribed to mRNA which is later translated to a specific protein molecule in the cell of an organism via transcription and translation respectively; and since the protein molecule is like the final product of a gene (DNA) expression in a cell, the mRNA is critical to the development of the actual protein molecule because it is the mRNA that decode the final genetic instruction encoded in the DNA for protein biosynthesis in the ribosome. (Proteins are the gene product of the cell). Because the mRNA molecule produced by the cell is complementary to its originator DNA molecule, the mRNA molecule will automatically bind to the original portion of the DNA strand from which it was copied or transcribed from; and this phenomenon is exploited in DNA microarray techniques to analyze several thousands of genes. The array or microarray is usually constructed from several DNA molecules or from the DNA-containing sample since it is the main genetic material that is transcribed to mRNA. The cDNA (which represent the mRNA in the cell) is labeled with a different fluorescent dye while the normal and abnormal samples are equally labeled with different dyes for easy characterization and identification at the end of the experimentation process. Labeled cDNA molecules bind or hybridize to their complementary DNA molecules immobilized or attached to the DNA microarray slide; and the intensity of the fluorescent for each of the spot on the DNA microarray slide is observed and measured via a specialized scanner connected to a computers system.

It is noteworthy that the amount of mRNA molecule bound to each site on the microarray plate or DNA microarray slide indicates the expression level of the various genes (which may range in several thousands) analyzed in the cell. In order words, the data so generated from the DNA microarray technique expressly show the gene expression in the cell; and each of the coloured spot on the microarray is unique and they each represent a particular gene (Figure 3). Comparing a normal gene with an abnormal gene allows molecular biologists and physicians to understand and elucidate the origin of several molecular diseases (e.g. cancer). When two cells (e.g. a normal cell and an abnormal cell) are co-hybridized in a DNA microarray technique, what happens is that both the normal cell and the abnormal cell (e.g. a cancer cell) will compete for the synthetic cDNA molecules on the DNA microarray slide; and since each of these cells are fluorescently-labeled and have different colours, it is easier for the researcher to elucidate which of the genes is expressed more than the other. Several techniques of DNA microarrays exist; and the type of DNA microarray experiment to undertake is dependent upon several factors which is normally determined by the researcher.

DNA microarray expression analysis, DNA microarray for mutation analysis and comparative genomic analysis are some of the various types of DNA microarray techniques available to undertake DNA microarray experimentation. The application of DNA microarray technique is boundless. DNA microarray technique is currently being used to evaluate the effects of toxins to cells; they have also been applied in the discovery of novel genes; DNA microarray is also applied in the discovery of novel drugs and other therapeutics; and this high-throughput technique is also applied in the diagnosis of diseases especially non-infectious molecular diseases such as cancer at the molecular level. Other applications of DNA microarray include: genotyping; estimating DNA copy number; measuring transcript abundance; identifying protein binding sites; measuring the decay rates of mRNA in the cell; and determining the subcellular localization of gene products in the cell of an organism.

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