BIO198L Gene Biotechnology Laboratory

1st Quarter SY 2015-2016

Southern Blot
Lomugdang, Fiord Jogardy B.1

1Student (s), Subject/Section, School of Chemical Engineering, Chemistry and Biotechnology, Mapua Institute of Technology

INTRODUCTION

Southern blotting is one of the central techniques in molecular

biology. First devised by E. M. Southern (1975) ,Southern blotting MATERIALS AND METHODS
results in transfer of DNA molecules, usually restriction fragments,
from an electrophoresis gel to a nitrocellulose or nylon sheet The following were the methods were used in this experiment:
(referred to as a membrane) , in such a way that the DNA banding I. Choose cut DNAs to fill the lanes in the gel
pattern present in the gel is reproduced on the membrane. During II. Cross link DNA to the filter.
transfer or as a result of subsequent treatment, the DNA becomes III. Set up the prehybridization.
IV. Select a radioactively labelled probe.
immobilized on the membrane and can be used as a substrate for
V. Denature the probe.
hybridization analysis with labelled DNA or RNA probes that VI. Add the probe to the roller bottle.
specically target individual restriction fragments in the blotted VII. Wash the filter.
DNA. In essence, Southern blotting is therefore a method for VIII. Place the filter in the X-ray cassette.
detection of a specic restriction fragment against a background
of many other restriction fragments. The restricted DNA might be
a plasmid or bacteriophage clone, Southern blotting being used to
conrm the identity of a cloned fragment or to identify an
interesting sub fragment from within the cloned DNA, or it might
be genomic DNA, in which case Southern blotting is a prelude to
techniques such as restriction fragment length polymorphism
(RFLP) analysis.

The objectives of this experiment are to choose genomic DNAs for

the Southern blot, select a fragment of DNA to use as a probe,
hybridize the Southern Blot with the probe and interpret the
hybridization pattern.

RESULTS & DISCUSSION

In the experiment, genomic DNAs were chosen to fill the lanes in

the gel. There were six genomic DNAs that were chosen. Then,
cross-linking pf the DNA to the filter were performed. The
permanent attachment of DNA to the nylon filter requires exposing
it to ultraviolet (UV) light. During gel electrophoresis, the DNA is
bound by ethidium bromide. When exposed to UV light, ethidium
Figure 1. Southern blotting.

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 BIO198L Gene Biotechnology Laboratory
1st Quarter SY 2015-2016

bromide emits an orange color, making RNA visible on the nylon

filter.

Next, pre-hybridization was performed. Hybridization analysis is

based on the principle that two polynucleotides will form a stable
hybrid by base-pairing if their nucleotide sequences are wholly or
partly complementary. A specific restriction fragment in a Southern
blot can therefore be detected if the membrane is probed with a
second, labelled DNA molecule that has the same, or similar,
sequence as the fragment being sought.

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 instance, 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.
Figure 3. X-ray image.
As seen from the figures above, figures 1 and 2, all sample DNA
were cut by the same restriction enzymes KpnI and AccI. The DNA
sequence for the -globin is in band 4.5 which is 1400 bp long.
The DNA of the normal person, sickle cell anaemia patient, -
thalassemia patients 2, 3 and 4 showed same band at 4.5 cm while
-thalassemia patient 1 showed no sign of the band. This must be
the result of the restriction enzymes detecting a restriction site in
the DNA sequence of -thalassemia patient number 1. The probe
used in this experiment was the -globin probe which detects the
sequence for the -globin gene. The experiment showed that -
thalassemia patient had the gene that contained a restriction site
for the restriction enzymes KpnI and AccI. This indicates that the
gene had a deletion mutation.

Figure 2. Ultraviolet image

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 BIO198L Gene Biotechnology Laboratory
1st Quarter SY 2015-2016

CONCLUSION

Southern blotting is a technique that enables a specific restriction

fragment to be detected against a background of many other
restriction fragments. It involves transfer of DNA fragments from
an electrophoresis gel to a nitro-cellulose or nylon membrane in
such a way that the DNA banding pattern present in the gel is
reproduced on the membrane. Hybridization probing is then used
to detect the restriction fragment that is being sought. The basic
methodology for Southern blotting has not changed since the
original technique was described in 1975, but modifications have
been introduced with the aim of speeding up the process and
achieving a more efficient transfer. Southern blotting has many
applications in molecular biology, including the identification of
one or more restriction fragments that contain a gene or other
DNA sequence of interest and in the detection of RFLPs used in
construction of genomic maps.

REFERENCES

Moffatt, B (2006). Course Notes Biology 208.Waterloo, University

of Waterloo.

Dale, J, & von Schantz, M (2003). From Genes to

Genomes.West Sussex: John Wiley & Sons Ltd.

C. Starr - R. Taggart: Cell Biology and Genetics, Brooks/Cole-

Thomson Learning, 2004.

Robert F. Weaver.5th ed. Molecular Biology, Published by

McGraw-Hill, 2012.

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