BB211: Cell and Molecular Biology

Dr Eve Lutz
Department of Bioscience
Recombinant DNA technology:
Lecture 2
Southern/Northern Analysis
Background reading:
Reference for this lecture please read the following:

Chapter 16
Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

Analysing complex nucleic acid

mixtures (DNA or RNA)
The total cellular DNA of an organism (genome) or the cellular
content of RNA is complex mixtures of different nucleic acid
sequences. Restriction digest of a complex genome can generate
millions of specific restriction fragments and there can be
several fragments of exactly the same size which will not be
separated from each other by electrophoresis.

Techniques have been devised to identify specific nucleic acids in

these complex mixtures

• Southern blotting - DNA

• Northern blotting - RNA

These techniques are not to be confused with Western blotting,

which is used to analyze PROTEINS which have been immobilized
on nitrocellulose/nylon filters. Proteins which have been
separated by polyacrylamide gel electrophoresis (PAGE) are
transferred to nitrocellulose/nylon filters and the filter is
probed with antibodies to detect the specific protein - similar to
the method used for expression library screening.

Southern blotting
This technique, devised by Ed Southern in 1975, is a commonly
used method for the identification of DNA fragments that are
complementary to a know DNA sequence. Southern hybridization,
also called Southern blotting, allows a comparison between the
genome of a particular organism and that of an available gene or
gene fragment (the probe). It can tell us whether an organism
contains a particular gene, and provide information about the
organization and restriction map of that gene.

In Southern blotting, chromosomal DNA is isolated from the

organism of interest, and digested to completion with a
restriction endonuclease enzyme. The restriction fragments are
then subjected to electrophoresis on an agarose gel, which
separates the fragments on the basis of size.

DNA fragments in the gel are denatured (i.e. separated into

single strands) using an alkaline solution. The next step is to
transfer fragments from the gel onto nitrocellulose filter or
nylon membrane. This can be performed by electrotransfer
(electrophoresing the DNA out of the gel and onto a
nitrocellulose filter), but is more typically performed by simple
capillary action.
 Fig 7-32, Lodish et al (4th ed.)
In this system, the denatured gel is placed onto sheet(s) of moist
filter paper and immersed in a buffer reservoir. A nitrocellulose
membrane is laid over the gel, and a number of dry filter papers
are placed on top of the membrane. By capillary action, buffer
moves up through the gel, drawn by the dry filter paper. It
carries the single-stranded DNA with it, and when the DNA
reaches the nitrocellulose it binds to it and is immobilized in the
same position relative to where it had migrated in the gel.

The DNA is bound irreversibly to the filter/membrane by baking

at high temperature (nitrocellulose) or cross-linking through
exposure to UV light (nylon).

The final step is to immerse the membrane in a solution containing

the probe - either a DNA (cDNA clone, genomic fragment, and
oligonucleotide) or RNA probe can be used. This is DNA
hybridization - in other words the target DNA and the probe
DNA/RNA form a 'hybrid' because they are complementary
sequences and so can bind to each other. The probe is usually
radioactively labeled with 32P (high energy β-particle emitter),
often by removal of the 5' phosphate of the probe with alkaline
phosphatase, and replacement with a radiolabeled phosphate using
γ-[32P] ATP and polynucleotide kinase. The membrane is washed to
remove non-specifically bound probe (see washing & stringency
conditions), and is then exposed to X-ray film - a process called
autoradiography. At positions where the probe is bound, β-
emissions from the probe cause the X-ray film to blacken. This
allows the identification of the sizes and the number of
fragments of chromosomal genes with strong similarity to the
gene or gene fragment used as a probe.

The principle of Southern blotting

What Southern blotting can tell us:

1. Whether a particular gene is present and how many copies

are present in the genome of an organism
2. The degree of similarity between the chromosomal gene and
the probe sequence
3. Whether recognition sites for particular restriction
endonucleases are present in the gene. By performing the
digestion with different endonucleases, or with
combinations of endonucleases, it is possible to obtain a
restriction map of the gene i.e. an idea of the restriction
enzyme sites in and around the gene- which will assist in
attempts to clone the gene.
 4. Whether re-arrangements have occurred during the cloning
process

An example of a Southern blot of human genomic clones

hybridised with a 1.5 kb cDNA probe

The size of this gene is ~117 kb and the mRNA transcript is ~4.8
kb - the gene is organised into 13 exons. Because of the large size
of the gene, several genomic clones, from YAC, PAC and cosmid
libraries, were isolated by hybridization screening with the cDNA
probe. In order to produce a partial restriction map of the gene,
the genomic clone cDNAs were digested with XhoI, HindIII,
EcoRI and BamHI individually, separated by agarose gel
electrophoresis and blotted onto nylon membranes. a) shows the
YAC clone digests (lanes 1-4), b) shows 2 different PAC clones
and their digests (PAC clone 1: lanes 1-4; PAC clone 2: lanes 6-9)
and c) shows 7 cosmid clones, each digested with BamHI. A cDNA
probe encoding the whole open reading frame (ORF) was labeled
with 32P and used to hybridize with these. Positive bands
correspond to DNA fragments containing exon sequences (a cDNA
probe will hybridize to fragments containing exon sequences).
Lanes containing the same size fragment for the same restriction
digest indicate that some clones overlap each other (contain the
same region of DNA). The sizes of the molecular weight markers
are shown alongside each blot.

Northern blotting
Northern blotting is a simple extension of Southern blotting -
and derives its name from the earlier technique. It is used to
detect cellular RNA rather than DNA. Initially, it was thought
that RNA would not bind efficiently to nitrocellulose, and other
modified materials were synthesized for use as a membrane.
However, it was then shown that when RNA was denatured, that
it would also bind efficiently to nitrocellulose. This means that
the RNA has to be unfolded into a linear strand before it will bind
efficiently to nitrocellulose. Chemicals such as formaldehyde and
methylmercuric hydroxide can be used to denature the RNA -
breaking down hydrogen bonding structure in the molecule. Alkali
is not used to denature the RNA - since RNA is degraded under
alkaline conditions.

Isolating RNA

RNA is extracted from the cells of interest - but precautions

must be taken to avoid degradation of the single-stranded RNA
by ribonuclease (RNase), which is found on the skin and on
glassware. Wear gloves, use specially treated plastics and
glassware to avoid accidentally introducing ribonuclease to
extraction prep. Addition of diethylpyrocabonate (DEPC) inhibits
ribonuclease activity and baking at high temperature destroys
ribonuclease activity (only useful for treating heat resistant
equipment, such as glassware).

Remember: it is important to maintain the integrity of RNA

molecules
RNA blots are most usually probed with cDNA fragments, but if a
single stranded probe (such as an oligonucleotide or a transcribed
RNA) is used - MAKE SURE IT IS COMPLEMENTARY TO THE
RNA SEQUENCE of the gene of interest.

What Northern blotting can tell us

1. Differential expression patterns of a particular gene

a) In which tissues it is expressed
b) If it is expressed during certain stages of development
c) If expression changes under differing
conditions/treatments of the cell
2. The quantity of the mRNA present

• Blots can be quantitated accurately by radioactive counting

3. Whether a genomic DNA probe has regions that are

transcribed

An example of a Northern blot exposed to film:

 Fig 7-33, Lodish et al (4th ed.)

Relative abundance of mRNA for β-globin in leukaemia cells that

are growing (UN) and in cells that have been induced to undergo
differentiation. The β-globin mRNA is produced when the cells
begin to differentiate.

Both Southerns & Northerns are exquisitely sensitive

Both Southern and Northern blots can detect very small amounts
of nucleic acids. Blots with both RNA and DNA from different
organisms (zoo blots) can inform us on the conservation of genes
among different organisms.

website last updated 20/2/03

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