Analysis of Gene Expression

ANALYSIS OF GENE EXPRESSION

The tools of biotechnology not only allow the study of gene structure, but also provide ways of analyzing the mRNA and protein products of gene expression.

A. Determination of messenger RNA levels

mRNA levels are usually determined by the hybridization of labeled probes to either mRNA itself or to cDNA produced from mRNA. [Note: Amplification of cDNA made from mRNA by retroviral reverse transcriptase (RT) is referred to as RT-PCR.]

1. Northern blots: Northern blots are very similar to Southern blots (see Figure 33.12), except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized to a radiolabeled probe. The bands obtained by autoradiography give a measure of the amount and size of particular mRNA molecules in the sample.

Figure 33.12 Southern blotting procedure. [Note: Nonradiolabeled probes are now commonly used.]

2. Microarrays: DNA microarrays contain thousands of immobilized ssDNA sequences organized in an area no larger than a microscope slide. These microarrays are used to analyze a sample for the presence of gene variations or mutations (genotyping) or to determine the patterns of mRNA production (gene expression analysis), analyzing thousands of genes at the same time. For genotyping analysis, the sample is from genomic DNA. For expression analysis, the population of mRNA molecules from a particular cell type is converted to cDNA and labeled with a fluorescent tag (Figure 33.22). This mixture is then exposed to a gene (DNA) chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene. The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the sample. DNA microarrays are used to determine the differing patterns of gene expression in two different types of cell (for example, normal and cancer cells; see Figure 33.22). They can also be used to subclassify cancers, such as breast cancer, to optimize treatment. [Note: Microarrays involving proteins and the antibodies or other proteins that recognize them are being used to identify biomarkers to aid in the diagnosis, prognosis, and treatment of disease based on a patient’s protein expression profile. Protein (and DNA) microarrays are important tools in the development of personalized medicine.]

Figure 33.22 Microarray analysis of gene expression using DNA (gene) chips. [Note: Protein chips are also used.] mRNA = messenger RNA; cDNA = complementary DNA.

B. Analysis of proteins

The kinds and amounts of proteins in cells do not always directly correspond to the amounts of mRNA present. Some mRNAs are translated more efficiently than others, and some proteins undergo posttranslational modification. When analyzing the abundance and interactions of a large number of proteins, automated methods involving a variety of techniques, such as mass spectrometry and two-dimensional electrophoresis, are used. When investigating one, or a limited number of proteins, labeled antibodies are used to detect and quantify specific proteins and to determine posttranslational modifications.

1. Enzyme-linked immunosorbent assays (ELISAs): These assays are performed in the wells of a plastic microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an antibody specific for the particular protein to be measured. The antibody is covalently bound to an enzyme, which will produce a colored product when exposed to its substrate. The amount of color produced is proportional to the amount of antibody present and, indirectly, to the amount of protein in a test sample.

2. Western blots: Western blots (also called immunoblots) are similar to Southern blots, except that protein molecules in the sample are separated by electrophoresis and blotted (transferred) to a membrane. The probe is a labeled antibody, which produces a band at the location of its antigen.

3. Detecting exposure to human immunodeficiency virus (HIV): ELISA and Western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV antibodies present in a patient’s blood sample. ELISAs are used as the primary screening tool, because they are very sensitive. Because these assays sometimes give false positives, however, Western blots, which are more specific, are often used as a confirmatory test (Figure 33.23). [Note: ELISA and Western blots can only detect HIV exposure after anti-HIV antibodies appear in the bloodstream. PCR-based testing for HIV is more useful in the first few months after exposure.]

Figure 33.23 Testing for HIV exposure by enzymelinked immunosorbent assays (ELISAs) and Western blots.

C. Proteomics

The study of the proteome or all the proteins expressed by a genome, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, is known as proteomics. The 20,000 to 25,000 protein-coding genes of the human genome translate into well over 100,000 proteins when posttranscriptional and posttranslational modifications are considered. Although a genome remains essentially unchanged, the amounts and types of proteins in any particular cell change dramatically as genes are turned on and off. [Note: Proteomics (and genomics) required the parallel development of bioinformatics, the computer-based organization, storage, and analysis of biologic data.] Figure 33.24 compares some of the analytic techniques discussed in this chapter.

Figure 33.24 Techniques used to analyze DNA, RNA, and proteins. ASO = allele-specific oligonucleotides. ELISA = enzymelinked immunosorbent assay; cDNA = complementary DNA; mRNA = messenger RNA.