Blotting & Probing Techniques Allow Visualization of Specific Target Molecules
المؤلف:
Peter J. Kennelly, Kathleen M. Botham, Owen P. McGuinness, Victor W. Rodwell, P. Anthony Weil
المصدر:
Harpers Illustrated Biochemistry
الجزء والصفحة:
32nd edition.p450-451
2025-10-23
60
Visualization of a specific DNA or RNA fragment (or protein, see later) among the many thousands of nontarget molecules in a complex sample requires the convergence of a number of techniques, collectively termed blot transfer. Figure1 illustrates the Southern (DNA), Northern (RNA), and Western (protein) blot transfer procedures. The first technique is named for the person who devised the technique, Edward Southern; the other names began as laboratory jargon, but are now accepted terms. These procedures are useful in determining how many copies of a gene are in a given tissue or whether there are any alterations in a gene (deletions, insertions, or rearrangements) because the requisite, initial gel electrophoresis step separates the molecules on the basis of size. Occasionally, if a specific base is changed and a restriction site is altered, these procedures can detect a point mutation. The Northern and Western blot transfer techniques are used to size and quantify specific RNA and protein molecules, respectively. A fourth technique, the Southwestern or overlay blot, which examines protein nucleic acid interactions or protein–protein interactions, respectively are variants of the Southern/Northern/Western blotting methods (not shown). In these last two techniques, proteins are separated by electrophoresis, blotted to a mem brane, renatured, and analyzed for an interaction with a particular DNA, or RNA sequence, or protein by incubation with a specific labeled nucleic acid probe (Southwestern) or protein probe (overlay assay) using either a labeled protein, or alternatively protein–protein interactions are detected using a specific antibody.
Fig. The blot transfer procedure. In a Southern, or DNA blot transfer, DNA isolated from a cell line or tissue is digested with one or more restriction enzymes. This mixture is pipetted into a well in an agarose or polyacrylamide gel and exposed to a direct electrical current. DNA, being negatively charged, migrates toward the anode; the smaller fragments move the most rapidly. After a suit able time, the DNA within the gel is denatured by exposure to mild alkali and transferred, via capillary action (or electrotransfer—not shown), to nitrocellulose or nylon paper, resulting in an exact replica of the pattern on the gel, using the blotting technique devised by Southern blot. The DNA is bound to the paper by exposure to heat or UV, and the paper is then exposed to the labeled DNA probe, which hybridizes to complementary strands on the filter. After thorough washing, the paper is exposed to x-ray film or an imaging screen, which is developed to reveal several specific bands corresponding to the DNA fragment(s) that were recognized (hybridized to) by the sequences in the DNA probe. The RNA, or Northern, blot is conceptually similar. RNA is subjected to electrophoresis before blot transfer. This requires some different steps from those of DNA transfer, primarily to ensure that the RNA remains intact, and is generally somewhat more difficult. In the protein, or Western, blot, proteins are electrophoresed and transferred to special paper that avidly binds proteins and then probed with a specific antibody or other probe molecule. (Asterisks signify labeled probes, either radioactive or fluorescent.) In the case of Southwestern blotting (see the text; not shown), a protein blot similar to that shown above under “Western” is exposed to labeled nucleic acid, and protein–nucleic acid complexes formed are detected by autoradiography or imaging.
All of the nucleic acid–based hybridization procedures discussed in this section depend on the specific base-pairing properties of complementary nucleic acid strands. Perfect matches hybridize readily and with stand high temperatures and/or low ionic strength buffer in the hybridization and washing reactions. Less than perfect matches do not tolerate such stringent conditions (ie, elevated temperatures and low-salt concentrations); thus, hybridization either never occurs or is disrupted during the washing step. Hybridization conditions capable of detecting just a single base-pair (bp) mismatch between probe and target have been devised.
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