Southern blotting is a technique that uses gel electrophoresis and hybridization probes to detect specific DNA sequences in genomic DNA. It involves digesting DNA with restriction enzymes, separating fragments by size via gel electrophoresis, transferring DNA to a membrane, and using a radioactive or other labeled probe to detect sequences that hybridize to the probe. Northern blotting adapts this technique to detect specific RNA sequences by separating RNA by size and hybridizing probes without restriction digestion. Both techniques allow detection and quantification of specific nucleic acid sequences.

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Northern & Southern
Hybridization
Avalon Garcia
02-20-04

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Introduction
 Concept: reannealing nucleic acids to identify sequence of
interest.
 Separates DNA/RNA in an agarose gel, then detects
specific bands using probe and hybridization.
 Hybridization takes advantage of the ability of a single
stranded DNA or RNA molecule to find its complement,
even in the presence of large amounts of unrelated DNA.
 Allows detection of specific bands (DNA fragments or RNA
molecules) that have complementary sequence to the
probe.
 Size bands and quantify abundance of molecule.

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Southern Blot: DNA-DNA*
Developed by Edwin Southern.
Uses gel electrophoresis together with hybridization
probes to characterize restriction fragments of genomic
DNA (or DNA from other sources, such as plasmids).
Identifies DNA with a specific base sequence.
Can be done to detect specific genes present in cells.

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Southern Steps
1. DNA to be analyzed is digested to completion with a
restriction endonuclease.
2. Electrophoresis to maximally separate restriction
fragments in the expected size range. A set of
standards of known size is run in one lane of the gel.
3. Blot fragments onto a nitrocellulose membrane.
4. Hybridize with the 32P probe.
5. Autoradiography.

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Step 2
Gel electrophoresis
 Separates DNA
fragments.
Soak gel in 0.5 M NaOH
 Converts dsDNA to ssDNA

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Step 3. Nitrocellulose
Blot
 Cover gel with nitrocellulose
paper…then…
 Cover nitrocellulose paper
with thick layer of paper
towels.
 Compress apparatus with
heavy weight.
 ssDNA binds to nitrocellulose
at same position it had on the
gel.
 Vacum dry nitrocellulose at
80C to permanently fix DNA
in place or cross link (via
covalent bonds) the DNA to
the membrane.

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Step 4. Hybridization
 Incubate nitrocellulose sheet
with a minimal quantity of
solution containing 32P-labeled
ssDNA probe.
 Probe sequence is
complementary to the DNA of
interest.
 Incubate for several hours at
suitable renaturation
temperature that will permit
probe to anneal to its target
sequence(s).
 Wash & dry nitrocellulose
sheet.

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Step 5. Autoradiography
 Place nitrocellulose sheet over
X-ray film.
 X-ray film darkens where the
fragments are complementary
to the radioactive probes.

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General Scheme for Southern Blot
http://homepage.smc.edu/colavito_mary/biology22/2

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Southern Application:
Diagnosis & detection of genetic diseases.
 Used to diagnose sickle cell-anemia.
 AT base change in the  subunit of Hb
Glu Val.
 Development of a 19 residue oligonucleotide probe
complementary to sickle-cell gene’s mutated segment.
 Probe hybridizes to DNA from homozygotes of sickle-cell
anemia but not from normal individuals.

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Northern Blot: RNA-DNA*(RNA*)
 Alwine adapted Southern's method for DNA to detect,
size and quantify RNA – 1977.
 No need to digest RNA with restriction enzymes.
 Use formaldehyde to break H-bonds and denature RNA
because single-stranded RNA will form intramolecular
base pairs and "fold" on itself.

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Northern Steps
1. Isolate RNA & treat with formaldehyde.
2. Electrophorese RNA in denaturing agarose gel (has
formaldehyde). Visualize RNA in gel using Ethidium
bromide stain and photograph.
3. Transfer single-stranded RNA to nitrocellulose or nylon
membrane. Covalently link RNA to membrane.
4. Incubate membrane (RNA immobilized on membrane)
with labeled DNA or RNA probe with target sequence.
5. Development.

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Step 1
Isolate RNA:
-To detect rare mRNA, isolate the poly A+ mRNA.
-RNA is both biologically and chemically more labile than
DNA. Thus eliminate RNases.
Step 2
Electrophoresis:
-Performed in formaldehyde agarose gel to prevent RNA
from folding on itself.
-Stain with EtBr to visualize the RNA bands.

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Step 3
-Transfer single-stranded RNA to nitrocellulose or nylon
membrane:
Traditionally, a nitrocellulose membrane is used,
although nylon or a positively charged nylon
membrane may be used.
Nitrocellulose typically has a binding capacity of
about 100µg/cm, while nylon has a binding capacity
of about 500 µg/cm. Many scientists feel nylon is
better since it binds more and is less fragile.
-Covalently link RNA to membrane:
UV cross linking is more effective in binding RNA to
the membrane than baking at 80C.

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Step 4 & 5
-Prehybridize before hybridization:
Blocks non-specific sites to prevent the single-stranded
probe from binding just anywhere on the membrane.
-Incubate membrane with labeled DNA or RNA probe with
target sequence:
Probe could be 32P, biotin/streptavidin or a
bioluminescent probe.
-Autoradiography:
Place membrane over X-ray film.
X-ray film darkens where the fragments are
complementary to the radioactive probes.

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Agarose gel & Membrane &
Autoradiography
Sobel, R., Dubitsky, A.,
Brickman, Y. (2002) Genetic
Engineering News 23, 2.

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Northern Application
 Northern blots are particularly useful for determining the
conditions under which specific genes are being
expressed, including which tissues in a complex
organism express which of its genes at the mRNA level.
 For instance:
When trying to learn about the function of a certain
protein, it is sometimes useful to purify mRNA from
many different tissues or cell types and then prepare a
Northern blot of those mRNAs, using a cDNA clone of
the protein of interest as the probe.
Only mRNA from the cell types that are synthesizing the
protein will hybridize to the probe.

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Summary
Southern
 DNA on membrane.
 Digest DNA.
 Convert dsDNA to
ssDNA.
 Probe with DNA or
RNA.
Northern
 RNA on membrane.
 No need to digest DNA.
 Denature “folded” RNA
with formaldehyde.
 Probe with DNA or RNA.

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References
Kornberg, A., Baker, T. A., DNA Replication. (2nd ed.) pp 15, W. H. Freeman &
Co. (1992)
Sobel, R., Dubitsky, A., Brickman, Y. (2002), Genetic Engineering News, 23, 2.
Southern, E. M., (1975) J. Mol. Biol. 98:503-517.
Voet, D., Voet, J. Biochemistry. (vol. 1, 3rd ed.), pp 110-112, John Wiley &
Sons Inc. (2004).
http://homepage.smc.edu/colavito_mary/biology22/2
www.colorado.edu/MCDB/MCDB2150Fall/notes99/99L17.html
www.uwp.edu/~higgs/Lect12mb.html
www.uwp.edu/~higgs/Lect13mb.html
www.sou.edu/biology/courses/bi342/Lect5.htm
www.med.unc.edu/pmbb/MBT2000/Northern%20Blotting.htm