Course: SAT 170 Introductory Biotechnology

Unit 7 Learning Module

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- Unit 7
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Page 3 of 5
DNA Technology Part 2 (continued)

This image illustrates the basic steps of the microarray technique.

Detection of Gene Expression

In an attempt to get a clearer picture on the mechanisms of how cells operate, gene expression studies are performed. The reason this is important is because not all genes are always expressed in the same way in different cells even though they are found in the same organism. As we discussed in unit 5, this means that the protein content or proteome of a particular organism is much more dynamic than that organism’s genome. mRNA is often used as a way to measure gene expression as it represents the product of transcription, which is the first step in gene expression (see Unit 4 for a review of this process). As we will see in this section, many of the DNA techniques are slightly modified for the detection or analysis of mRNA content.

The FISH method is one method that can be modified to detect gene expression by isolating mRNA rather than chromosomes for attachment to the FISH microscope slide. mRNA specific, labeled probes can then be used in much the same way, but will indicate the presence of RNA molecules rather than DNA sequences as previously described.

DNA Microarrays are sometimes referred to as gene chips since they are just analyzing the DNA that is being expressed in a cell sample at a certain time. These microarrays are created by applying small amounts of single stranded DNA molecules to a small glass microscope slide. There are thousands of different DNA samples, each with a unique sequence of DNA, that are applied to the same slide. Each sample is applied to a specific region or spot of the slide. This process of impregnating the slide with these DNA samples is done by a high-speed robotic arm which contains a number of tiny pins, each immersed in a specific DNA sample. This robotic arm is known as an arrayer. mRNA can then be extracted from a particular source of cells (such as a certain tissue) and can be copied into cDNA molecules as previously described. These cDNA molecules are then labeled and incubated with the microarray to allow them to attach to any complementary sequences they may find on the microarray. After a wash to remove any unbound cDNA, the slide can then be examined for fluorescence of the labeled cDNA molecules. The level of fluorescence can not only be used as an indicator of gene expression but it can also indicate the level of gene expression. For example, no fluorescence indicates that the gene was not expressed in the original cell/tissue sample, while moderate or bright fluorescence indicates moderate to high expression of a particular gene, respectively.

This image illustrates the arrangement of the small DNA fragments that are applied by the microarrayer to a microscope or glass slide for a DNA microarray. | A researcher examines a DNA microarray using the computer generated images that show different levels of fluorescence dependent on the different levels of gene expression in the sample being tested. | An image of the commercially available “Gene Chip” used for DNA microarrays.

Northern Blotting is another technique used to evaluate the expression of certain genes within an organism. This technique follows the same basic steps as the Southern Blot, with the exception that mRNA, rather than DNA samples are the ones that are collected and analyzed.

This image illustrates the basic steps of the Northern Blot technique. | This figure illustrates gels collected from the various electrophoresis methods that we have been discussing including the Western Blot from our last unit and the Northern Blot from this unit.