Blotting Techniques

Mazen Al Zaharna
MSc Biological Sciences- Medical Technology
Medical Technology Dep.
The Islamic University- Gaza
Southern Blotting
Southern Blotting
 Developed by E.M. Southern in
1975.
 A technique used in molecular
biology to check for the presence
of a particular DNA sequence in a
DNA sample.
Flow chart of Southern hybridization
Preparing the samples and running the gel

Southern transfer & Fixing DNA onto membrane

Isotope
Probe preparation Non-isotope

Prehybridization

Hybridization

Post-hybridization washing

Signal detection
Preparing the samples and running the
gel
 Extraction of DNA
 DNA must first be fragmented into
small pieces that can migrate through
an agarose gel matrix.
 Restriction enzymes are used to
fragment the DNA
Preparing the samples and running the gel
DNA Digestion
 Restriction enzymes recognize
specific DNA sequences in DNA and
cleave the DNA at these restriction
sites.
 Digestion with a given restriction
enzyme produces a set of fragments
that are easily separated by agarose
gel electrophoresis.
Preparing the samples and running the gel
DNA Digestion

The enzyme EcoRI cutting DNA at its recognition sequence

Preparing the samples and running the gel
Electrophoresis
 Nucleic acids are negatively charged at a
neutral pH
 This allows their migration through an
electric field
 Agarose is a highly porous polysaccharide
that acts as a sieve, allowing the
fragments of DNA to be separated
according to length.
Preparing the samples and running the gel
Electrophoresis
Preparing the samples and running the gel
Denature the DNA
 Denature DNA with an alkaline
solution such as NaOH.
 Double stranded becomes single-
stranded.
 Single strands are ready to be
transferred to a solid support
Southern Transfer & Fixing DNA

 Transfer the DNA from the gel to a

solid support.
 Baking the membrane at 80°C for 2 h
in a vacuum oven.
 Or expose to ultraviolet
Probe Preparation, Prehybridization &
Hybridization
 A labeled probe is prepared which is
complementary for the sequence we
are looking for
 Prehybridization to block sites where
probe can bind on the membrane
 Hybridization
Post-hybridization washing
 Following hybridization, the blot must
be washed to remove unassociated
and nonspecifically annealed probe
from the blot.
Detection
Steps in Southern Blotting
DNA extraction
Disease gene

Fragments of
DNA digestion DNA appear
as a smear
Gel electrophoresis

Paper towels
Chromatography Denaturation of patient’s DNA in gel
paper support
Gel in
Nylon filter
Southern blot NaOH
Gel
10x SSC
Blot dismantled
Autoradiography
X ray film
Hybridisation:
Radioactive probe Stringency washes
added to filter
cassette
filter filter
Uses of Southern Blotting Technique
 Identify mutations, deletions, or
rearrangements that alter the integrity of a
specific gene,
useful in the prognosis of certain types of
cancer
 Tool for molecular cloning, providing a
mechanism for localization of specific
sequences
Uses of Southern Blotting Technique

 The DNA blot can also be used to assess

the relative copy number of a specific
gene.
Useful in detecting gene amplification.
 Southern blotting may be used to confirm
the specificity of the test reaction product.
 To search for a homologous gene different
organisms
Southern Blotting as a Diagnostic
Method
 Restriction fragment length polymorphism
(RFLP) analysis was one of the early
methods to diagnose point mutations
implicated in genetic diseases
 The change in the size of detected fragments
with a gene-specific probe signals the
presence of mutation in the analyzed gene
 Has been applied to the diagnosis of
hemophilia A, Sickle cell anemia and others
Southern Blotting as a Diagnostic
Method
 PCR has replaced the Southern
blotting
 Cystic fibrosis, Duchenne muscular
dystrophy, sickle cell anemia
thalassaemia, and others, are now
diagnosed by polymerase chain
reaction (PCR).
Genomic and Plasmid DNA Analyses

 Does a particular genomic locus or region of

plasmid DNA contain a sequence of interest?
Where does it reside?
 Techniques:
 Restriction enzyme digestion
 Agarose gel electrophoresis
 Southern blot
Genomic and Plasmid DNA Analyses

 How many genomic loci contain a particular

sequence of interest, or how many copies of
that sequence does a genome contain?
 Technique:
Southern blot
The Northern Blot
The flow chart of Northern hybridization
Prepare RNA samples and run RNA gel

Northern transfer

Isotope
Probe preparation
Non-isotope

Prehybridization

Hybridization

Post-hybridization washing

Signal detection
 Allows identification of specific messenger
RNA sequences within a mixture of RNA
molecules.
 The final signal achieved on the blot is
proportional to the number of specific
sequences present,
allowing for a quantitative analysis of gene
expression.
Differences between Southern & Northern

 RNA rather than DNA is separated by size on gel

 Cutting by nucleases before electrophoresis
unnecessary.
 Although RNA is single stranded, it has a
tendency to bend back on itself and form base-
paired loops, hairpins, and other secondary
structures.
 Denaturing agents (e.g., formamide) must be
added to the electrophoresis buffer to prevent the
formation of secondary structures
Differences between Southern &
Northern
RNA Paranoia
 RNA paranoia is very important from start
to finish.
 The work area should be cleaned with
RNase inhibitors.
 Gloves should be changed if non-RNase-
free items have been touched (e.g., your
hair, your face, your arm, notebook
paper).
Uses of Northern Blotting
 Northern blots can be used to assess
different levels of expression from a
particular gene.
 For defining post-transcriptional
modification such as:
splicing and poly(A) addition,
Gene Expression (Transcription) Analyses

 What is the size of a specific gene

transcript?
 Technique:
Northern blot
Gene Expression (Transcription) Analyses

 Is a gene of interest expressed

(transcribed)?
 Technique:
Northern blot
Gene Expression (Transcription) Analyses

 Is transcription of a gene altered

(increased or decreased) under different
conditions?
 Technique:
Northern blot
Real-time PCR (for more quantitative
comparison)
 Immunoblotting
(Western Blotting)
The Flow Chart Of
Immunoblotting
Electrophorese samples

Transfer proteins from gel to membrane

Blocking

Addition of 1o Ab, washing

Addition of 2o Ab, washing

Detection
Uses of Immunoblotting
 Immunoblotting is used to identify
specific protein in a mixture
Uses of Immunoblotting
Dot and slot blots

 Provide a quick and simple way to

determine the amount of an antigen in a
sample without performing electrophoresis
first.
 Proteins are deposited onto the membrane
 Probe with the same chromogenic or
luminescence protocol as the western blot.
Dot and slot blots
 Provides a mean of measuring the
abundance of specific proteins without the
need for gel electrophoresis,
 It does not, however, provide information
regarding the size of the fragments.
Proteins
 In which cellular structures or organelles do
specific proteins reside?
 Techniques:
Cell fractionation
Immunoblotting
Proteins
 What is the molecular mass of a specific
protein? Is it post-translationally modified?
 Technique:
Immunoblotting
Example for Uses of Blotting Techniques
 Suppose a student was studying a newly
identified gene, X, from cows.
 The student then asks three basic questions
as part of a research project:
1. Do sheep also have gene X on their chromosomes?
2. Do cows express gene X in their brain tissue?
3. Is the protein product of gene X found in the cow's
blood plasma?
 Blotting experiments can answer all three of
these questions.
Do sheep also have gene X on their chromosomes?

 A Southern (DNA) blot will answer the first

question.
 DNA from a sheep and performed the Southern
blotting technique with a probe complementary
to that gene.
 If the sheep's DNA also contains gene X, there
should be a fragment on the nitrocellulose
 In other words, the labeled probe will bind to any
fragment from the blotted sheep DNA that
contains gene X, allowing the student to detect
the presence of gene X in sheep.
Do cows express gene X in their brain tissue?

 To answer the second question, a

Northern (RNA) blot would be used.
 The student would isolate RNA from the
cow's brain tissue and run it out on the
gel.
 The same DNA probe used for Southern
would then be used to detect whether the
RNA that represents gene X expression is
present in the brain.
Is the protein product of gene X found in the cow's
blood plasma?

 To answer the third question, the student

would use a Western (protein) blot.
 This requires the use of an antibody that
specifically reacts with the protein coded
for by gene X.
 The student first obtains plasma from the
cow and uses standard biochemical
techniques to isolate the proteins for
analysis.
 These proteins can then be run out
on a gel and transferred to
nitrocellulose.
 The proteins can then be probed with
the labeled antibody.
 If the product of gene X is in the
plasma, it will bind with the labeled
antibody and can thus be detected.
References
 Current Protocols Essential Laboratory
Techniques (2008)
 Molecular Diagnostics (2006)
 Medical Biomethods Handbook (2005)
Thank You