MLAB 2378 Fundamentals of Molecular Diagnostics Links

MLAB 2378 Fundamentals of Molecular Diagnostics

Unit 7 Notes- Resolution and Detection of Nucleic Acids

Brief Overview

Once nucleic acids are extracted and the concentration and purity are determined procedures are performed to characterize the nucleic acids. The simplest system is electrophoresis. DNA is negatively charged and will migrate toward the positive pole, the anode, when exposed to an electrical charge. The migration pattern will be determined by the size and charge. When characterizing fragments of DNA and RNA media is selected to separate fragments on the basis of numbers of base pairs (bp) in the fragment. (Note, kilobase (kb) is a thousand base pairs). Fragments with a large number of base pairs will migrate more slowly than smaller fragments. The method can be made more selective by altering the test system. For example, if agarose gel is used varying the concentration of the gel will change the electrophoretic pattern. A 2% gel will separate out fragments from small to very large whereas higher gel concentrations will give a better resolution of smaller fragments. Gels routinely used range from 2-5%. Higher or lower concentrations are not practical. Polyacrylamide is another popular gel but acrylamide is a potent neurotoxin and must be handled with great care. Be able to compare and contrast the use of agarose and polyacrylamide gels.

Familiarize yourself with the basics of the 3 electrophoretic methods: pulse field gel, polyacrylamide gel and capillary electrophoresis. Be able to compare the three methods so far as the performance, advantages and disadvantages of each . I would encourage you to make a chart.

The buffering system is an essential element of electrophoresis. The purpose of the buffer is to carry the current and protect the samples during electrophoresis. Modifying the buffer system with buffer additives is a method used to affect migration. DNA and RNA require different buffering additives due to the nature of their structure.

Electrophoresis equipment has NOT changed much over the many years that it has been used. Gel, buffer, electrodes, and current are still the primary components. Teaching laboratories prefer that students make their own gels as part of the learning process to see exactly what the gel is composed of. It also allows the students to utilize math skills so that they can determine the volume of gel needed and the amounts of "ingredients" necessary to create a gel of a certain size AND thickness. A comb is used to create the wells where the nucleic acids and loading dye will be added.

Dyes which specifically associate with nucleic acids are added for detection of the bands produced after electrophoresis. Ethidium bromide (EtBr), SyBr green and silver stain are the most popular.

Ethidium Bromide is visualized under UV light of 300 nm and visible light at 500 nm. This dye is VERY carcinogenic and great care must be used when handling it. Most laboratories capture the image of the bands on gel with a camera.

SyBr green is 25-100 times more sensitive than EtBr and is not as toxic. The disadvantage is that special optical filters are required for detection of SyBr green.

Silver stain was originally developed for visualization of proteins. It is not an intercaltor but it is a biohazard. The technique is more complicated as the color development must be watched carefully so the reaction can be stopped at the appropriate time.

I encourage you to create a chart to compare the purpose, advantage and disadvantage of each dye.