Methodology Of Southern Blot Analysis

4.1. PREPARATION OF DNA FOR SOUTHERN BLOTTING Most basic techniques for purification of DNA produce material appropriate for Southern analysis. Standard Southern blot protocols recommend the use of 10 |g of DNA when analyzing single-copy genes (6). However, when the amount of DNA is limiting, smaller quantities can be used without compromising the signal by altering the geometry of the sample well during electrophoresis (decreasing the width of the well will increase the intensity of the final signal) (7). In situations where multiple copies of the gene are present or if the gene constitutes a high percentage of the DNA, such as when plasmid DNA containing the gene of interest is being analyzed, the quantity of DNA can be reduced dramatically to as little as 200 ng. Under optimal conditions, rare sequences such as single-copy genes can be detected when 10 |g of genomic DNA are analyzed (6). DNA that is to be analyzed by Southern blot must first be fragmented into small pieces that can migrate through an agarose gel matrix. Restriction enzymes are bacterial enzymes that recognize specific DNA sequences (four to six nucleotides long) in DNA and cleave the DNA at these restriction sites. Digestion of genomic DNA with a given restriction enzyme produces a reproducible set of fragments that are easily separated by agarose gel electrophoresis. In order to determine which restriction enzyme(s) to use, it is helpful to know which restriction sites are present within and around the gene of interest. Generally, when evaluating the presence or copy number of a particular gene, one should avoid using restriction enzymes that cut the gene of interest into a large number of small fragments. Ideally, the gene of interest should be cleaved into a few fragments (one to three) that range in size from 1.0-10.0 kilobases (kb). A different approach might be desirable when evaluating the integrity of a specific gene. The average gene contains many restriction sites, and cleavage of the gene with a particular restriction enzyme produces a distinct number of fragments of a defined size. Mutations, deletions, or rearrangements occurring within a gene could result in a disruption of the normal nucleotide sequence, possibly altering the number of restriction sites within the gene or altering the size of the restriction fragments produced. Such a change in the size pattern of DNA fragments produced by enzymatic cleavage is referred to as a restriction fragment length polymorphism (RFLP). A detailed restriction map of a gene is generated by cleaving the DNA with several restriction enzymes separately and then performing a Southern analysis of the fragmented DNA with a probe for the gene of interest. Restriction maps are useful for identifying subtle differences between homologous genes. Hundreds of restriction enzymes are commercially available (Invitrogen [Carlsbad, CA], Sigma [St. Louis, MO], New England Biolabs [Beverly, MA]), and manufacturers typically provide the proper buffer necessary for digestion as well as instructions for the quantity of enzyme, temperature, and duration of reaction required for thorough digestion. One unit of a restriction enzyme is defined as the amount required to cleave 1.0 |g of DNA in 1 h. Complete cleavage of the DNA is essential for Southern blotting, especially when single-copy genes are being analyzed. A small aliquot (0.5-1.0 |g) of the digested DNA sample can be subjected to agarose gel elec-trophoresis and stained with ethidium bromide to determine whether enzymatic digestion of the DNA is complete (see Figs. 1 and 2). Thorough digestion of plasmid DNA is evidenced by total disappearance of the uncut plasmid and the appearance of specific bands. Digestion of genomic DNA is confirmed by the presence of a continual ladder of fragmented DNA along with distinct bands, usually in the lower portion of the gel. These bands are produced from enzymatic fragmentation of repetitive elements within the DNA and are characteristic of the restriction enzyme. After fragmentation, the DNA is typically concentrated by phenol/chloroform extraction, ethanol precipitation, and resuspended in a small volume of elec-trophoresis buffer in preparation for electrophoretic separation (see Table 1).

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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