Molecular biology: Blotting / Hybridization techniques
Author: Dr Henriëtte van Heerden
Licensed under a Creative Commons Attribution license.
What is blotting / hybridization?
surface. Homologous DNA from another source will
Blotting of nucleic acid is the central technique for
hybridize to the immobilized DNA. Non-homologous
hybridization studies.
Double stranded DNA will
DNA will not bind / attach. This is the basis of DNA
denature or separate at high temperatures into single
probe techniques. A probe is a piece of DNA with a
strands. When the temperature is lowered, the two
specific nucleic acid sequence that is labelled with a
strands will anneal because of the base pairing
marker which allows identification and quantification.
interaction between the complementary strands. If
similar,
All blotting procedures begin with a standard process
complementary strands from different sources will
called gel electrophoresis when DNA, RNA, or proteins
anneal. This is called hybridization to indicate that each
are loaded on to an agarose or acrylamide gel and
strand of DNA came from a different source.
separated on the gel through an electric field. Two
nucleotide
sequences
are
identical
or
types of gels are commonly used: agarose gels and
acrylamide
gels.
Transfer
is
initiated
when
nitrocellulose or nylon membrane is laid on top of the
gel and biological molecules are transferred from the
gel to the membrane. Hybridization / blotting is a
technique in which biological molecules (DNA, RNA or
protein) are immobilized onto a nylon or nitrocellulose
membrane. A probe (a piece of nucleic acid with
identical and specific sequence to the organism or
gene of interest) can then hybridize (join) to the
biological molecules (DNA, RNA or protein) with an
identical
DNA probe joins together (hybridizes) with target DNA
blotted on a membrane.
sequence
on
the
membrane.
The
hybridization between the blotted DNA and probe is
visualized by labelling the probe in some way.
The single strands that hybridize to one another to form
double stranded DNA are homologous (have similar or
identical nucleotide sequences). This high specificity of
base
pairing
interaction
between
complementary
strands of DNA can be used to locate specific
nucleotide sequences in a sample. The DNA from one
source can be immobilized by attachment to a solid
What are the different types of hybridization?
Blotting is the technique in which nucleic acids or
proteins are immobilized onto a solid support, generally
nylon or nitrocellulose membranes. There are different
blotting procedures depending on the type of molecule
being
transferred.
When
DNA
fragments
are
transferred the procedure is called a Southern blot,
separate proteins. Shorter molecules move faster and
named after the person who first developed it, Edward
migrate further than longer ones because the shorter
Southern. With Northern blotting, RNA molecules are
molecules migrate more easily through the pores of the
transferred
gel. The different sized molecules form distinct bands
and
with
Western
blotting,
protein
molecules are transferred.
What
are
the
on the gel which can be visualized using stains.
basic
blotting/hybridization
Transfer is initiated when the gel is retrieved from the
procedures?
electrophoresis apparatus and the nylon / nitrocellulose
All blotting procedures begin with a standard process
membrane is laid on top of the gel. The objective now
called gel electrophoresis. During this step, DNA, RNA,
is to transfer the bands of molecules found in the gel to
or
or
the membrane. The molecules are immobilized (fixed)
polyacylamide gel (that functions like a molecular
on the membrane. Short fragments of DNA that have a
sieve) and are then run through an electric field.
complementary nucleotide sequence to the molecule
proteins
are
loaded
on
to
an
agarose
being analyzed are normally used as probes in
Southern and Northern blots. Proteins/antigens that
react with the proteins/antibodies being analyzed are
used as probes in a Western Blot.
Example of hybridization
The reverse
A schematic example of an agarose gel electrophoresis
apparatus
line
blotting (RLB)
technique
was
developed using a PCR assay that amplifies a region
of a detectable marker, so that the resulting PCR
product is labelled. Pathogen-specific probes are
blotted onto a membrane in ‘lines’ and then hybridized
with the labelled PCR products, which are applied in
perpendicular lines (the technique is known as the
reverse line blot because the probes are immobilized
on the membrane rather than the PCR products, and
the PCR products are labelled rather than the probes).
Hybridization will only occur between a matching
amplified product and specific probe, and hybridized
Polyacrylamide Gel Electrophoresis (PAGE) a) The gel is
poured vertically between two glass plates. b.) Protein bands
are separated on the basis of relative molecular weight and
visualized with stains. (Adapted from Seidman and Moore.
Basic Lab Methods for Biotechnology. Prentice Hall, New
Jersey)
PCR products can then be detected via the label. RLB
thus combines PCR amplification with a hybridization
step making the technique up to 1000 fold more
sensitive than PCR alone. The RLB has the additional
advantage of allowing the analysis of multiple samples
against
multiple
probes
to
enable
simultaneous
Two types of gels are commonly used to separate
detection of and discrimination between pathogenic
molecules according to size and/or charge: agarose
organisms present in the samples. Development of
gels are used to separate DNA and RNA, and
each assay involves (i) careful primer and probe
polyacrylamide gel electrophoresis (PAGE) is used to
design, based on literature and sequence database
searches, which are critical to the success of the
assay; and (ii) bench-top evaluation, using known
samples, controls and dilution series, to confirm
sensitivity, specificity and reproducibility. The assay
takes about one and half working days to complete;
about 4 h for the PCR and 6 h for the RLB, including a
total of 4 h 'hands-on' time.