12.2 Visualizing and characterizing dna, rna, and protein  (Page 28/16)

Learning objectives

• Explain the use of nucleic acid probes to visualize specific DNA sequences
• Explain the use of gel electrophoresis to separate DNA fragments
• Explain the principle of restriction fragment length polymorphism analysis and its uses
• Compare and contrast Southern and northern blots
• Explain the principles and uses of microarray analysis
• Describe the methods uses to separate and visualize protein variants
• Explain the method and uses of polymerase chain reaction and DNA sequencing

The sequence of a DNA molecule can help us identify an organism when compared to known sequences housed in a database. 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. 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. Analysis of protein signatures can reveal the identity of an organism or how a cell is responding during disease.

The DNA and proteins of interest are microscopic and typically mixed in with many other molecules including DNA or proteins irrelevant to our interests. Many techniques have been developed to isolate and characterize molecules of interest. 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. For example, many pathogens, such as the bacterium Helicobacter pylori , which causes stomach ulcers , can be detected using protein-based tests. 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.

Molecular analysis of dna

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. Some of these methods do not require knowledge of the complete sequence of the DNA molecule. 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.

Nucleic acid probing

DNA molecules are small, and the information contained in their sequence is invisible. 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. Probes can be used to identify different bacterial species in the environment and many DNA probes are now available to detect pathogens clinically. For example, DNA probes are used to detect the vaginal pathogens Candida albicans , Gardnerella vaginalis , and Trichomonas vaginalis .