16 - Chapter 16. METHODS USED FOR STUDYING GENE EXPRESSIO N...

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Unformatted text preview: Chapter 16. METHODS USED FOR STUDYING GENE EXPRESSIO N A wide array of powerful analytical techniques has accumulated during the past 40 years and these have revolutionized the study of biology and the understanding of basic mechanisms concerning the genetic control of living systems. In the following sections, a very brief overview of some of the important techniques is provided. Readers seeking a more comprehensive treatment should consult texts on molecular biology or biochemistry. The present description is aimed at explaining the basis of how some of the methods are applied in some areas of understanding animal function. A. Basic techniques 1. Electrophoretic separation: Charged molecules migrate in electric fields and this is combined with a gel of some kind to minimize spreading of molecules by diffusion. For nucleic acids, aqueous gels made from agarose stabilize the separation medium (the electric field is established between an anode and a cathode through the buffer in an intervening gel) and the concentration of the agarose in the gel determines the extent to which the movement of the nucleic acid is restricted. Gels of suitable agarose content restrict the migration of larger molecules to a greater extent than that of smaller molecules which enables separation of like-charged molecules on the basis of their size. For separating nucleic acids, agarose is melted and dispersed in a buffer of a pH that ensures that the nucleic acids carry an appreciable negative charge so they will migrate electrophoretically towards the anode. The molten agarose is poured into a slab format which includes slots/wells near the cathode end that are formed for applying samples and a DNA mixture of known sizes ( a ladder ), used for calibration. The samples and ladder are loaded into the wells then separated by the applied electric field. After adequate separation, DNA in the gel is stained and visualized with a suitable dye reagent. Fluorescent dyes which change their emission characteristics upon binding to DNA are widely used and visualization usually employs UV-wavelength light with suitable filters and some form of image capture - a film or digital camera (Figure 16- 1). If required, segments of the gel containing the size-separated DNA can be cut from the slab and the DNA can be recovered. Other applications depend upon being able to transfer the separated nucleic acids from the gel onto a membrane replica (a "blot"), the DNA or RNA is then covalently attached to the membrane and it is then used for further analyses, typically using hybridization with nucleic acid probes (described below). 2007 version page 112 Figure 16-1. DNA electrophoresis. DNA has been separated, on the basis of size, in a gel of agarose after being loaded into the wells visible at the top and at mid-gel....
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16 - Chapter 16. METHODS USED FOR STUDYING GENE EXPRESSIO N...

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