Southern Blot

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A Southern blot is a method routinely used in molecular biology for detection of a specific
DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-
separated DNA fragments to a filter membrane and subsequent fragment detection by probe
hybridization. The method is named after its inventor, the British biologist Edwin Southern.[1]
Other blotting methods (i.e., western blot, northern blot, eastern blot, southwestern blot) that
employ similar principles, but using RNA or protein, have later been named in reference to
Edwin Southern's name. As the technique was eponymously named, Southern blot should be
capitalized as is required for proper nouns, whereas names for other blotting methods should
not.
Contents
[hide]
 1 Method
 2 Result
 3 See also
 4 References
 5 External links
[edit] Method
1. Restriction endonucleases are used to cut high-molecular-weight DNA strands into
smaller fragments.
2. The DNA fragments are then electrophoresed on an agarose gel to separate them by
size.
3. If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel
may be treated with an acid, such as dilute HCl, which depurinates the DNA
fragments, breaking the DNA into smaller pieces, thus allowing more efficient
transfer from the gel to membrane.
4. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution
(typically containing sodium hydroxide) to denature the double-stranded DNA. The
denaturation in an alkaline environment may improve binding of the negatively
charged DNA to a positively charged membrane, separating it into single DNA
strands for later hybridization to the probe (see below), and destroys any residual
RNA that may still be present in the DNA. The choice of alkaline over neutral transfer
methods, however, is often empirical and may result in equivalent results.[citation needed]
5. A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or
below, depending on the direction of the transfer) the gel. Pressure is applied evenly
to the gel (either using suction, or by placing a stack of paper towels and a weight on
top of the membrane and gel), to ensure good and even contact between gel and
membrane. Buffer transfer by capillary action from a region of high water potential to
a region of low water potential (usually filter paper and paper tissues) is then used to
move the DNA from the gel on to the membrane; ion exchange interactions bind the
DNA to the membrane due to the negative charge of the DNA and positive charge of
the membrane.
6. The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours
(standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet
radiation (nylon membrane) to permanently attach the transferred DNA to the
membrane.
7. The membrane is then exposed to a hybridization probe—a single DNA fragment
with a specific sequence whose presence in the target DNA is to be determined. The
probe DNA is labelled so that it can be detected, usually by incorporating
radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some
cases, the hybridization probe may be made from RNA, rather than DNA. To ensure
the specificity of the binding of the probe to the sample DNA, most common
hybridization methods use salmon or herring sperm DNA for blocking of the
membrane surface and target DNA, deionized formamide, and detergents such as SDS
to reduce non-specific binding of the probe.
8. After hybridization, excess probe is washed from the membrane (typically using SSC
buffer), and the pattern of hybridization is visualized on X-ray film by
autoradiography in the case of a radioactive or fluorescent probe, or by development
of color on the membrane if a chromogenic detection method is used.
[edit] Result
Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that
this fragment contains DNA sequence that is complementary to the probe.
The transfer step of the DNA from the electrophoresis gel to a membrane permits easy
binding of the labeled hybridization probe to the size-fractionated DNA. It also allows for the
fixation of the target-probe hybrids, required for analysis by autoradiography or other
detection methods.
Southern blots performed with restriction enzyme-digested genomic DNA may be used to
determine the number of sequences (e.g., gene copies) in a genome. A probe that hybridizes
only to a single DNA segment that has not been cut by the restriction enzyme will produce a
single band on a Southern blot, whereas multiple bands will likely be observed when the
probe hybridizes to several highly similar sequences (e.g., those that may be the result of
sequence duplication). Modification of the hybridization conditions (for example, increasing
the hybridization temperature or decreasing salt concentration) may be used to increase
specificity and decrease hybridization of the probe to sequences that are less than 100%
similar.
[edit] See also

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