DNA+microarray+(GeneChip)

ALLAN DONAHOE

DNA microarrays were introduced in 1995, and has been significantly utilized and improved immensely since its discovery. The first reported microarray was carried out where 45 cDNA probes were spotted on a glass slide, the DNA was immobilized and the ensuing microarray was used for gene expression analysis. Within one year 1000 probes were arrayed, to get a sense of how rapidly this technology improved. Since its invention there have been three advancements that significantly improved its effectiveness. Most substantially is the miniaturization of the spots for better sensitivity and analysis of more genes as well as the use of fluorescence for detection and the use of glass as a rigid solid support.3 DNA microarray technology has immeasurably changed the field of transcriptomics, the study of the expression of all genes in a particular organism of interest. Simply put, DNA microarrays involve depositing a plethora of DNA segments, ranging up to several hundred thousand, in a precise array on a solid surface, most commonly a coated glass surface to be analyzed by the ability of the target DNA to hybridize to the array. The binding of DNA is shown by Figure 1, which explains how and which types of modified can DNA bind to the microarray.


 * Figure 1. ** Immobilization of DNA to surfaces. (A) Unmodified DNA is randomly immobilized to surfaces meaning that some DNA strands can participate in hybridization (I) while other cannot (II). (B) Immobilization of DNA using end modifications (I) can also result in intra chain bonds (II). (C) Molecular organization of end modified probes directly immobilized to the active groups on the solid support (I) or displaced from the surface using a linker (II) or a dendrimeric linker (III).

The first step of a DNA microarray is the isolation of mRNA from cells or tissues from the organism of interest. The mRNA is then reverse-transcribed into complementary DNA and labeled with a cyanine fluorescing dye, either red (CY3) or green (CY5). The cDNA is allowed hybridize to the DNA on the microarray, as shown in Figure 1, and the cDNA that is not tightly bound is washed. The intensity of the fluorescence is then identified using an automated scanning-laser microscope, with the relative intensity indicating how much cDNA, and therefore the amount of mRNA, is bound to the particular complementary DNA in each microarray spot. Generally, the fluorescent cDNA reverse-transcribed from the isolated mRNA is mixed with fluorescent cDNA from a differently colored reference sample. From this it can be determined if the amount of RNA expressed from a particular gene from the experimental organism by the color fluoresced. If the gene expression levels increased the color is observed to be the same as labeled sample cDNA and if the expression levels decreased the color is the same as the reference cDNA. If there was no change in the expression levels relative the reference, the color is the mixing of two dyes utilized. For example, if the sample cDNA was labeled red and the reference cDNA green, no change in expression levels is observed to be yellow. This use of reference cDNA labeled a different color allows for identification of gene expression with high precision.1,2. Figure 2 is used to summarize the GeneChip. Figure 2. summarizes how the GeneChip functions. The target stands bind to the corresponding sequences on the GeneChip, the genes are identified and quantified by the computer program [9]. Figure 3 shows an example of a the fluorescence detected using a DNA microarray, but however a more complex two-dimensional clustering of microarray data.4

** Figure 3. ** 2D clustering of microarray through adult human hippocampus samples and differentially expressed genes across hippocampal subdivisions (ANOVA, P, 0.01 BH-corrected, top 5,000 genes), with selected enriched The use of DNA microarrays to analyze gene expression provides numerous amounts of information. The expression of all the genes of an organism can be determined as well as simply a small change in gene expression in a specific pathway. An example of a commonly used technique is cluster analysis, which is extremely useful in understanding the gene expression in an essential metabolic pathway. This is done by analyzing genes that are coordinately regulated, that is they are turned off and on in sync towards a specific influence. Additionally DNA microarrays can be used to monitor the progression of DNA replication in a cell, as described in an ensuing research experiment, or to quickly identify disease-causing microbes.1,2,3 Research conducted by a group constructing an anatomically comprehensive atlas of the adult human brain transcriptome is an example of how powerful using DNA microarrays can be.4 This technology allowed these researches to generate and analyze a transcriptional atlas of the adult human brain by a comprehensive microarray profiling of approximately 900 neuroanatomically precise subdivisions in two individuals. From the analysis of differential gene expression and gene co-expression relationships, they were able to conclude that brain-wide variation results precisely from the distribution of cell classes such as neurons, oligodendrocytes and microglia. This research determined that genes involved in synaptic transmission affect the local neighborhood relationships between anatomical subdivisions. The most important result of this experiment was that the spatial topography of the neocortex is determined by its molecular topography, that is the closer the two cortical regions the more similar their transcriptomes. The compilation of this data resulted in a transcriptional basis for neurogenetic studies of normal and abnormal brain function. 4 Another research experiment conduced that uses DNA microarrays is the direct reprogramming of somatic to determine its promotion by maternal transcription factor Glis1.5 The basis of this research involved induced pluripotent stem cells which are generated from somatic cells, although inefficiently, by transgenic expression of three transcription factors, called OSK as a group. The major finding of this research is that the transcription factor Glis1 (Glis family zinc finger 1) enhances the formation of iPSCs from human and mice fibroblasts when expressed along with OSK. The DNA microarray analyses indicated that Glis1 promotes multiple reprogramming pathways and therefore effectively promotes the direct reprogramming of somatic sells within iPSC generation.5 A final research experiment analyzed the spatiotemporal regulation of cell-cycle genes by SHORTROOT links patterning and growth.6 This paper explores the fact that molecular mechanisms for the regulation of formative cell divisions remains not very well understood. //Arabidopsis// was utilized, where the formative divisions generating the root ground tissue are controlled by SHORTROOT (SHR) and SCARECROW (SCR). This experiment uses a unique DNA microarray, which is a chromatin immunoprecipiation-based microarray. This array data along with the transcriptional effects of SHR and SCR allowed them to conclude that SHR regulates the spatiotemporal activation of genes involved in cell division. The results also indicate that correct pattern formation is accomplished by transcriptional regulation of specific cell cycle genes in a cell-type and developmental-stage- specific context.6 In 2013 the Journal of Clinical Microbiology published an article that used GeneChip analysis to detect //M. tuberculosis// drug resistant species. This study is the first large scale evaluation of GeneChip in basic-level TD laboratories. The GeneChip assay was based on the genotyping of the genes associated with drug resistance in //M. tuberculosis// by reverse hybridization. GeneChip analysis has many advantages over other techniques that can be used in similar situations, including tests can be completed within 6 hours and GeneChip is based on testing for nucleic acids. The team was able to prove that conventional methods had a false positive of about 20-30% when testing for rifampin-resistant TB and MDR-TB. The team concluded that GeneChip can be a more effective, rapid, safe, and cost-beneficial alternative to conventional DST [6]. A research team in Taiwan used GeneChip analysis to detect the gene expression of circulating tumor cells in the blood of patients with colorectal cancer. Colorectal cancer (CRC) in the most common tumor in the world. Survival rates are low, primarily because of the consistently poor prognosis of patients who have advanced stage CRC. Patients with CRC showed overexpression rate of nine genes was greater than 90% of the CRC patients, and GeneChip arrays was able to pick up on the over expression coupled with other techniques. The team concluded that GeneCling CRC Enzymatic Gene Chip Detection Kit is easy, fast, and convenient to operate for detecting gene expression of CTCs from peripheral blood. The new method can be used to help diagnose patients with CRC and help speed up the treatment process [7]. In 2013 a research team in China used GeneChip analysis to help diagnose hereditary spastic paralegias, which previously had been a time consuming and costly process. The recent advances in molecular-level interaction and detection technology are upgrading the clinical diagnostics, particularly GeneChip analysis. GeneChips or DNA microarrays can be efficiently used in clinical diagnosis. The team demonstrated the suitability of 96-plex GoldenGate assay for detecting site mutations of HSPs, with a consistency of 99.0%. The high successful performance of this GoldenGate assay makes it a useful technique for preliminary genetic screening for HSP patients and it may be used in clinic in the future. It is expected that GeneChip assays will play an increasingly important role in disease diagnosis in the near future [8].

[1] Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. & Walter, P. //Molecular Biology of the Cell: 5th Edition//. Garland Science, Taylor and Francis Group, LLC: New York. 2008 [2] Voet, D., Voet, J.G., Pratt, C.W. //Fundamentals of Biochemistry – Life at the Molecular Level: 4th edition//. John Wiley & Sons, Inc: New Jersey. 2013. [3] Dufva, Martin. Fabrication of high quality microarrays. //Biomedical Engineering (22)// 173-184.2005. [4] Hawrylycz, M.J. et. al. An anatomically comprehensive atlas of the adult human brain transcriptome. //Nature (489) 391-399//. 2012. [5] Maekawa, M. et. al. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. //Nature (474), 225-230//. 2011. [6] Sozzani, R. et. al. Spatiotemporal regulation of cell-cycle genes by SHORTROOT links patterning and growth. //Nature (466) 128-134.// 2010. [6] Luo, Yingying, Juan Du, Zixiong Zhan, Chong Chen, Junling Wang, Yiqiao Hu, Zhengmao Hu, Kun Xia, Beisha Tang, and Lu Shen. "A Diagnostic Gene Chip for Hereditary Spastic Paraplegias." //Brain Research Bulletin// 97 (2013): 112-18. Web. [7] Pang, Y., H. Xia, Z. Zhang, J. Li, Y. Dong, Q. Li, X. Ou, Y. Song, Y. Wang, R. O'brien, K. M. Kam, J. Chi, S. Huan, D. P. Chin, and Y. Zhao. "Multicenter Evaluation of Genechip for Detection of Multidrug-Resistant Mycobacterium Tuberculosis." //Journal of Clinical Microbiology// 51.6 (2013): 1707-713. Web. [8] Wu, Chan-Han, Fu-Yen Chung, Jia-Yuan Chang, and Jaw-Yuan Wang. "Rapid Detection of Gene Expression by a Colorectal Cancer Enzymatic Gene Chip Detection Kit." //Biomarkers and Genomic Medicine// 5.3 (2013): 87-91. Web. [9] "Infographics | Tolpa Studios." //Infographics | Tolpa Studios//. TOLPASTUDIOS, INC., n.d. Web. 12 Oct. 2014.