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Nucleic Acid Hybridizations

Southern hybridization - hybridization of a probe to filter bound DNA; the DNA


is typically transferred to the filter from a gel (A technique developed by E. M.
Southern in 1975 for the detection of a specific DNA sequence)
Northern hybridization - hybridization of a probe to filter bound RNA; the RNA
is typically transferred to the filter from a gel
Heating the DNA solution above a characteristic temperature can separate the two strands
of a double helix. That temperature is called the melting temperature, abbreviated Tm .
Above the Tm, a DNA is mostly or all single-stranded; below the Tm, it is mostly doublestranded. For a natural DNA, the Tm depends primarily on its G+C content. Because a G
C base pair has three hydrogen bonds and an AT pair only has two, nucleic acid double
helices with a high G+C content have a higher Tm than do those with a greater proportion
of A+T. The Tm is not an exact property: It depends on the solvent conditions. For
example, a high concentration of salt (such as NaCl) raises the Tm of a DNA duplex,
because the positive Na+ ions shield the negative charges on the phosphodiester
backbone from repelling each other. Likewise, certain organic solvents can cause the
negative charges on the phosphates to repel more strongly; these solvents lower the Tm of
a DNA double helix.
What happens if two nucleic acids are partly complementary and partly different? In this
case, some stretches of the two strands may form base pairs while others don't. The two
molecules can be manipulated so that they form a hybrid or separate. The conditions
favoring the formation of duplex nucleic acid are low temperature (below the Tm), high
salt, and the absence of organic solvents. The latter two conditions raise the Tm of the
hybrid duplex so that the DNA would remain more double-stranded. On the other hand,
higher temperatures (closer to the Tm of the hybrid) lower salt, and the presence of
organic solvents would tend to push the two strands of the DNA apart. The term
stringency sums up these variables: The more stringent the conditions, the more likely
partially complementary sequences are to be forced apart. Conversely, less stringent
hybridization conditions mean that the two strands need not be so complementary to form
a stable helix.

Southern and Northern blot hybridization


Southern blot hybridization refers to the detection of specific DNA fragments that have
been separated by gel electrophoresis.
After the electrophoresis the separated DNA fragments are denatured and transferred to a
nitrocellulose (or nylon) membrane sheet by blotting. In the blotting the gel is supported
on a sponge in a bath of buffer solution. Buffer is sucked through the gel and the sheet by
paper towels stacked on top of the nitrocellulose sheet.

The buffer denatures the DNA and transfers the single stranded fragments from the gel to
the surface of the sheet, where they adhere firmly.
The nitrocellulose sheet containing the bound single-stranded DNA fragments is pealed
off the gel and placed in a sealed plastic bag or a box together with buffer containing
labelled DNA probe specific for the target DNA sequence.
The sheet is exposed to the probe under conditions favoring hybridization.
After the hybridization, the sheet is removed from the bag, washed thoroughly to remove
unhybridized probes and viewed using autoradiography or ultraviolet light depending on
the labels used (radioactive of fluorescent).
An adaptation of Southern blotting is Northern blotting, in which RNA molecules are
electrophoresed through the gel instead of DNA.