DNA often must first be isolated from bodily samples through chemical extraction methods before a DNA probe can be used to identify pathogens. While these techniques are valuable for diagnosis, their direct use on sputum and other bodily samples may be problematic due to the complex nature of these samples. The DNA sample being probed must also be denatured to make it single-stranded so that the single-stranded DNA probe can anneal to the single-stranded DNA sample at locations where their sequences are complementary. In either case, the DNA probe must be labeled with a molecular tag or beacon, such as a radioactive phosphorus atom (as is used for autoradiography) or a fluorescent dye (as is used in fluorescent in situ hybridization, or FISH), so that the probe and the DNA it binds to can be seen ( Figure 12.13). The DNA probe may be synthesized chemically by commercial laboratories, or it may be created by cloning, isolating, and denaturing a DNA fragment from a living organism. If researchers have a portion of the sequence of DNA for the gene of interest, they can design a DNA probe, a single-stranded DNA fragment that is complementary to part of the gene of interest and different from other DNA sequences in the sample. To screen a genomic library for a particular gene or sequence of interest, researchers must know something about that gene. For example, DNA probes are used to detect the vaginal pathogens Candida albicans, Gardnerella vaginalis, and Trichomonas vaginalis. Probes can be used to identify different bacterial species in the environment and many DNA probes are now available to detect pathogens clinically. How does a researcher isolate a particular stretch of DNA, or having isolated it, determine what organism it is from, what its sequence is, or what its function is? One method to identify the presence of a certain DNA sequence uses artificially constructed pieces of DNA called probes. Nucleic Acid ProbingĭNA molecules are small, and the information contained in their sequence is invisible. Before the advent of rapid DNA sequencing, these methods were the only ones available to work with DNA, but they still form the basic arsenal of tools used by molecular geneticists to study the body’s responses to microbial and other diseases. Some of these methods do not require knowledge of the complete sequence of the DNA molecule.
In this subsection, we will outline some of the basic methods used for separating and visualizing specific fragments of DNA that are of interest to a scientist. In addition, an increasing number of highly specific and accurate DNA amplification-based identification assays can now detect pathogens such as antibiotic-resistant enteric bacteria, herpes simplex virus, varicella-zoster virus, and many others. For example, many pathogens, such as the bacterium Helicobacter pylori, which causes stomach ulcers, can be detected using protein-based tests. These methods were originally developed for research purposes, but in many cases they have been simplified to the point that routine clinical use is possible. Many techniques have been developed to isolate and characterize molecules of interest. The DNA and proteins of interest are microscopic and typically mixed in with many other molecules including DNA or proteins irrelevant to our interests. Analysis of protein signatures can reveal the identity of an organism or how a cell is responding during disease. Comparing protein signatures-the expression levels of specific arrays of proteins-between samples is an important method for evaluating cellular responses to a multitude of environmental factors and stresses. The sequence can also tell us something about the function of a particular part of the DNA, such as whether it encodes a particular protein. The sequence of a DNA molecule can help us identify an organism when compared to known sequences housed in a database. Explain the method and uses of polymerase chain reaction and DNA sequencing.Describe the methods uses to separate and visualize protein variants.Explain the principles and uses of microarray analysis.Compare and contrast Southern and northern blots.Explain the principle of restriction fragment length polymorphism analysis and its uses.Explain the use of gel electrophoresis to separate DNA fragments.Explain the use of nucleic acid probes to visualize specific DNA sequences.By the end of this section, you will be able to:
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