Expression Pattern of Genes Using DNA Microarrays

Paradigm shift in science always goes hand-in-hand with technological breakthroughs. The program by which a complex body develops from a single fertilized cell requires the understanding of the spatial and temporal regulation of gene transcription in response to external and internal cues. In 1995, Patrick O. Brown and his colleagues at Stanford University introduced the DNA microarray technique for analyzing global expression of genes (Schena et al., 1995). The yeast cell cycle was one of the first biological processes to be interrogated by this new technique. The commercial yeast microarray contains 6200 genes covalently linked on glass slides as about 100 ^m dots in grids of 96 or 384 spots. Each gene is amplified by polymerase chain reaction (PCR) and the purified DNA is used for printing the microarrays. Microarray technology (Figure 6.14) is fully automated, carried out by robots to increase throughput and minimize error. The microarray experiment measures the relative abundance of messages in a cell, revealing a picture of a cell's transcriptome. It has been applied successfully to identify induction and repression of genes during specific cellular processes. The first gene expression analysis examined the genes that are regulated in a cell-cycle dependent manner in yeast (Spellman et al., 1998) by comparing the relative abundance of mRNA as cells progressed through the cell cycle. Messenger RNA was harvested at defined time points to capture different phases of the cell cycle and converted into complimentary DNA (cDNA) using appropriate primers and the enzyme reverse transcriptase in the presence of red (Cy5) and green (Cy3) fluorescently labeled nucleotide precursors. Labeled cDNA (Cy3) from an asynchronous culture (control) was mixed with labeled cDNA from the synchronous culture (Cy5) and hybridized to a DNA microarray containing the yeast genes. The cDNA sequences representing individual transcripts hybridized specifically to corresponding gene sequences on the array. The fluorescence associated with each spot was quantitated using a microscope (microarray reader) that illuminates each spot with a laser beam and measures the fluorescence associated with each dye separately to estimate the relative abundance of the transcript by the ratio of the red to green fluorescence. About 800 genes were found to be cell cycle regulated, which could be clustered into groups, whose expression correlated with specific phase of the cell cycle. For example, about 100 genes were found to be co-regulated in G1. Many of these genes function in establishing cellular polarity and initiation of bud growth, providing a molecular link between budding with the position of the cell in G1. The microarray technique has become a standard tool to understand the mechanism of disease incidence and progression and has been applied successfully in cancer.

Was this article helpful?

0 0

Post a comment