Southern Blot Analysis
Southern transfer, DNA blot, & Gel-transfer
hybridization
•Southern blotting was named after Edward M.
Southern, a British biologist who developed this
procedure in 1975.
•It is a biochemical technique often used in Molecular
Biology and Biochemistry.
•It is a method for transferring DNA fragments from
agarose gel, used for electrophoresis, to a nitrocellulose
or nylon derivative membrane.
•The technique is used to detect the presence of specific
DNA fragments in a complex mixture.
•The DNA detected can be a single gene, or it can be
part of a larger piece of DNA such as a viral genome.
•The key to this method is hybridization.
Southern Blotting Steps:
Restriction of total chromosomal DNA: DNA, either a single
gene or of the total genome is digested with a restriction
enzyme, often restriction endonuclease.
DNA fragments separation using gel electropherosis:
Restriction is followed by separation, using gel electrophoresis
(usually an agarose gel is used). The purpose of gel
electrophoresis is to separate DNA fragments.
Depurination: DNA is partially depurinated with dilute HCL
which promotes higher efficiency transfer by breaking down
fragments into smaller pieces.
Denaturation: The next step involves denaturing the DNA with
sodium hydroxide (NaOH) or another alkaline/basic chemical
substance. The DNA is now in single stranded, rather than in
the double helix.
Neutralization: DNA is then neutralized with NaCl to prevent re-
hybridization before adding the probe.
Blotting: The single-stranded DNA is transferred to a nitrocellulose
membrane, which is placed on the gel.
Weight is put on top of the membrane; this is usually a stack of
paper towels. The purpose of the paper towels over the filter is for
DNA to participate in a process known as capillary action, paper
towels absorb the alkaline chemical substance, and thus binds to
the single-stranded DNA.
Prehybridization blocking
Buffer binds to areas on the blot not occupied by sample DNA.
Blocks the empty sites from being bound during hybridization.
Hybridization: Membrane fixed with the DNA fragments is
placed in hybridization bottle with radioactive hybridization
probe, made of single-stranded DNA or sometimes RNA. The
hybridization probe is tagged using radioactivity,
fluorescence, or a chromogenic dye. Enzymes can also be
attached to the probe as horseradish peroxidase or Alkaline
phosphatase.
Washings: Following this step, any excess probe is cleaned off
the membrane.
Cross linking of DNA: The membrane is put under ultraviolet
light or heated until baked – this is an important step to
permanently fix the DNA onto the nitrocellulose membrane.
The blot is made permanent by:
•Drying at ~80°C
•Exposing to UV irradiation
Examining the results (detection):
• Using radioactive or fluorescent probe: Then,
hybridization patterns are viewed on x-ray film by a
process known as autoradiography, if a radioactive or
fluorescent probe is used.
• Using chromogenic probe: if using chromogenics, the
membrane is color-developed for detection.
Chromogenic detection of biotin labelled probe hybrid
Streptavidin (a biotin binding tetrameric protein) is conjugated to
alkaline phosphatase, which facilitates chromogenic detection.
Alkaline phosphatase cleaves the substrate (BCIP-T 5-bromo-4-
chloro-3-indolyl phosphate, p-toluidine salt) which is
supplemented with the enhancer chromogen NBT (nitro blue
tetrazolium). This results in the forma
Applications of the Southern Blot.
➢To identify a single gene among thousands of fragments of DNA and to
detect sequences of DNA in an organism’s genome.
➢Used in gene discovery and gene mapping.
➢To analyze the genetic patterns in an organism’s DNA.
➢To identify gene mutations, deletions, duplications, and gene
rearrangements involved in diseases.
➢To determine the number of copies of a particular DNA sequence
presented in the genome of an organism.
➢It can also be used to measure the molecular weight of a restriction
fragment.
➢To identify related DNA sequences in the genome and to determine if
there is a gene family (a group of similar genes).
➢To detect certain cancers and genetic diseases, such as:
•Monoclonal leukemia populations
•Sickle cell mutations
➢Used in DNA fingerprinting, genetic engineering, & forensic science as:
•Paternity testing
•Personal identification
•Sex determination
•Species exclusion
Advantages
❖Effective way to detect a specific DNA sequence in a large,
complex sample of DNA
❖More suitable than various other tests to analyze long
stretches of DNA
❖Can be used to semi-quantify the amount of DNA present
❖Used as a definitive test when identifying genetically modified
organisms
❖Cost effective than DNA sequencing
Disadvantages
❖Complex and laborious
❖Time consuming and cumbersome
Depurination Solution Denaturation Solution
0.25 M conc. HCl 20g NaOH
58.4g NaCl
20 x SSC (Standard Sodium Neutralization Solution
Citrate) 78.8g Tris base
175.3g NaCl 87.6g NaCl
88.2g Sodium Citrate Ajust pH to 7.4
Pre-hybridization Solution
15 ml Deionized formamide
9 ml SSC (20X)
3 ml Denhaurdant’s Solution
1.5 ml SDS (10%)
Prehybridization blocking reagents
Blocking protein: minimizes non specific binding of probe to
membrane
✓Casein (milk protein)
✓Denhaurdt’s solution (BSA, Ficoll)
DNA hybridization: The formation of a duplex between two
complementary sequences is called hybridization.
There are two important features of hybridization:
•The reactions are specific-the probes will only bind to targets
with a complementary sequence.
•The probe can find one molecule of target in a mixture of millions
of related but non-complementary molecules.
Factors affecting the hybridizing signals
•Amount of genomic DNA
•Proportion of the genome that is complementary to the
probe
•Size of the probe
•Labeling efficiency of the probe
•Amout of DNA transferred to membrane
•Presence of denaturing agents (formamide)
Stringency: It includes the conditions of hybridization that
control the specificity of binding between two single strand
portions of nucleic acid (usually probe and immobilized target
sequence)
•Increased temperature or decreased ionic strength, increases
stringency
•Only duplex with near perfect sequence complementarity
can exist at high stringency.
Troubleshooting
Poor signal
➢ Probe specificity too low
➢ Inadequate transfer buffer
➢ Not enough target DNA
➢ Transfer time too short
➢ Inefficient transfer system
➢ Probe concentration too low
➢ Incomplete denaturation of probe or target DNA
➢ Stringent wash
➢ Hybridization time too short
➢ Inappropriate membrane
Spotty Background
➢ Unincorporated nucleotides not removed from labeled probe
➢ Particles in hybridization buffer
➢ Agarose dried on membrane
➢ Baking or UV crosslinking when membrane contains high salt
Troubleshooting (High background)
➢ Insufficient Blocking
➢ Membrane being dried out during hybridization or washing
➢ Membranes adhered during hybridization or washing
➢ Bubbles in hybridization bag
➢ Walls of hybridization bag collapsed on to membrane
➢ Not enough wash solution
➢ Hybridization temperature too low
➢ Labeled probe molecules are too short
➢ Probe Concentration too high
➢ Inadequate prehybridization
➢ Probe not denatured
➢ Not enough SDS in wash solution
