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L4 Blotting and Nucleic Acid Hybridization Techniques

Specific objectives
By the end of this lecture, you should be able to:
1. Explain the process of nucleic acid hybridization
2. Describe the following blotting and hybridizaition techniques.
(a) Southern blot hybridization
(b) Northern blot hybridization
(c) Dot blot hybridization
3. Explain how chromosomes are separated by electrophoresis.
4. State three applications of Northern blotting and hybridization.

Introduction
Although after cloning the aim may be to obtain the gene sequence, it is usually not sensible to begin
sequencing straight away. For example, if a 20 kb fragment of genomic DNA has been cloned in a
replacement vector, and the area of interest is only 2 kb in length, much effort would be wasted by
sequencing the entire clone. It is essential to determine which parts of the original clone contain the
region of interest. This is done by using blotting techniques.

Nucleic acid hybridization is the formation of a double-stranded molecule by base-pairing between

complementary or homologous polynucleotide.
It is an extremely sensitive detection technique used to pick out specific DNA sequences from complex
mixtures. Usually a single pure sequence is labelled with 32P and used as a probe.
It is carried out as follows:
 The probe is denatured before use so that the strands are free to base-pair with their complements.
 The DNA to be probed is also denatured, and is usually fixed to a supporting membrane made
from nitrocellulose or nylon.
 Hybridization is carried out in a sealed plastic bag or tube at 65-68oC for several hours to allow the
duplexes to form.
 The excess probe is then washed off.
 The degree of hybridization can be monitored by counting the sample in a scintillation
spectrometer, or preparing an autoradiogram, where the sample is exposed to X-ray film.

Southern blotting
 Southern blotting is the transfer of single-stranded, restricted DNA fragments, separated in an
agarose gel, to a nylon or nitrocellulose membrane which is then analyzed by hybridization to
radioactive or biotinylated single-stranded DNA or RNA probes. The hybrids are detected by
autoradiography or a colour change, respectively.
 It is used for detecting fragments in the agarose gel that are complementary to a given RNA or
DNA sequence.
 The main blotting procedures are:
 Blotting of nucleic acids from gels
 Dot and slot blotting
 Colony and plaque blotting.
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How Southern blotting is performed

 DNA is denatured with base to that the resulting single-stranded DNA can bind to the
nitrocellulose membrane, forming the Southern blot.
 Large DNA fragments, greater than 10 kb, require a longer transfer time than short fragments.
Therefore to allow uniform transfer of a wide range of DNA fragment sizes, the electrophoresed
DNA is exposed to a short depurination treatment by 0.25 M HCl followed by alkali.
 Depurination has the following effects:
 It shortens the DNA fragments by alkaline hydrolysis at depurination sites
 It denatures the fragments prior to transfer ensuring that they are in the single-stranded state
and accessible for probing.
 The gel is equilibrated in neutralizing solution prior to blotting.
 The agarose gel is mounted on a filter paper wick that dips into a reservoir containing transfer
buffer.
 The hybridization membrane is sandwiched between the gel and a stack of paper towels which
serves to draw the transfer buffer through the gel by capillary action.
 The DNA molecules are carried out of the gel by the buffer flow and immobilized on the
membrane on either nitrocellulose or supported nylon membranes. The latter membrane is
preferable because it has a greater binding capacity for nucleic acids in addition to high tensile
strength.
 DNA is fixed to the nitrocellulose or nylon membrane by oven baking at 80 oC. Due to the
flammable nature of nitrocellulose, nylon membrane is baked in a vacuum oven???????????.
 Ultraviolet cross-linking is an alternative method for DNA fixation. The method is based on the
formation of crosslinks between a small fraction of the thymine residues in the DNA and
positively-charged amino groups on the surface of nylon membranes.
 DNA polymerase is applied to the probe DNA in presence of radioactive DNA precursors to make
it radioactive.
 The membrane with the fixed DNA is placed in a solution of labelled (radioactive or non-
radioactive) RNA, single-stranded DNA, or oligodeoxynucleotide (probe).
 Wherever the probe encounters a complementary DNA fragment, it hybridizes to it.
 After the hybridization reaction, the membrane is washed to remove unbound radioactivity and
regions of hybridization are detected autoradiographically by placing the membrane in contact
with X-ray film.
 Southern blotting is extremely sensitive and can be used to detect minute amounts of DNA.
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Southern Hybridization

Locating the position of a cloned gene on a large DNA molecule

Problem
Southern hybridization is appropriate for locating cloned genes on small DNA molecules such as from
most plasmids, bacteriophages and viruses. It cannot be used to locate cloned genes on larger DNA
molecules such as cloned eukaryotic genes on chromosomal DNA molecules. Restriction mapping
becomes very complicated with molecules more than about 250 kb in size. Other techniques have to
used.
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Separating chromosomes by gel electrophoresis

In order to find out which chromosome carries a gene of interest, one has to use a special type of
Southern hybridization, involving not restriction fragments but intact chromosomal DNA molecules,
separated by a novel type of gel electrophoresis.
In conventional electrophoresis only molecules within a certain size range can be separated because
the difference in migration rate becomes increasingly small for larger molecules. In practice, molecules
larger than about 50 kb cannot be resolved efficiently by standard gel electrophoresis.

The limitations of standard gel electrophoresis can be overcome if a more complex electric field is
used. One of the systems used is called orthogonal field alternation gel electrophoresis (OFAGE).
Instead of being applied directly along the length of the gel, the electric field now alternates between
two pairs of electrodes, each pair set at an angle of 45o to the length of the gel. The result is a pulsed
field, with the DNA molecules in the gel having continually to change direction in accordance with the
pulses.

As the two fields alternates in a regular fashion the net movement of the DNA molecules in the gel is
still from one end to the other, in more or less a straight line. However, with every change in field
direction each DNA molecule has to realign through 90o before its migration can continue. A short
molecule can realign faster than a long one and progresses towards the bottom of the gel more quickly.
In this technique, the resolving power of the gel is increased dramatically and molecules up to several
thousand kb can be separated. The size range includes the chromosomal molecules of many eukaryotes
e.g. yeast, several filamentous fungi and protozoans e.g. malaria parasite.
(see figure below of gel showing chromosomes)
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Northern blotting
 Northern blotting is a technique analogous to Southern blotting used for transferring RNA from an
agarose gel to a nitrocellulose membrane on which it is bound covalently.
 Once bound to the membrane, the RNA can be hybridized to a complementary (radioactive)
single-stranded DNA or RNA probe.
 As for Southern analysis, hybridization bands are located by autoradiography.
 The name of the technique “Northern blot” is colloquial and not after the person who developed
the technique.
 Northern hybridization is used to determine the amount and size of intact RNA, separated on a
denaturing agarose gel.
Applications
(a) Determination of hybridization patterns in mRNA samples.
(b) Determining which regions of a cloned DNA fragment will hybridize to a particular mRNA.
(c) The technique is often used in measuring transcript levels during expression of a particular
gene. For example,
(i) After cloning a gene from the Petunia one may want to find out which tissue -leaves, stems, roots,
flowers, or seeds transcribes the gene to the greatest extent. Northern blot technique could be used as
follows:
 Collect mRNAs from all five tissues
 Electrophorese them
 Then Northern blot them
 Hybridize a radioactive probe made from the cloned petunia gene to the Northern blot
 Then carry out autoradiography.
The tissue that contains the highest concentration of the specific mRNA will hybridize best and will
give the most intense band on the autoradiograph
(ii) Suppose that in a bacterial vector you have cloned a plant gene that codes for a photosynthesis
protein, and you wish to find out if this gene is active in roots and other nonphotosynthetic tissue, how
would you go about this?
 Get the bacterial vector containing the gene of interest into the cell type which you wish to
test. This can be accomplished by one of several means such as “shooting” the vector into the
cell or producing transformed plants.
 Assuming that you also have a resistance gene in the vector, you can select for that gene.
 Once the tissue is expressing the resistance gene, indicating possible integration of the vector
into the plant chromosome, you can assay for the protein of interest or for production of its
mRNA.
 The quick way would be to do a Northern blot on the root’s enzymes and the resultant pieces
separated on an electrophoresis gel, a pattern of bands can be visualized by hybridization
methods.

Dot blotting
This is a technique in which small spots or ‘dots’ of nucleic acid are immobilized on a nitrocellulose or
nylon membrane and hybridization then carried out as for Northern or Southern blots.
The technique is particularly useful in obtaining quantitative data in the study of gene expression. An
example of a dot blot is shown below.
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Western blotting
This technique involves the transfer of electrophoretically separated protein molecules to membranes.
The membrane is then probed with an antibody to detect the protein of interest, in a similar way to
immunological screening of plaque lifts from expression libraries.