Hybridization and blotting

Learning outcomes
• identify principle, components, and types of the nucleic acid
hybridization.
• Describe different approaches of probe labelling and detection.
• Recognize and differentiate between different blotting methods like;
Southern blot, northern blot, and western blot.
• Identify colony hybridization and fluorescence in situ hybridization
and their applications
 Hybridization
• It is the pairing of nucleic acid sequences (DNA
and RNA) that are complementary to produce a
double strand (duplex).
• It is used to detect the presence of target
sequences within a complex mixture of DNA or
RNA molecules .
• It consists of the DNA or RNA of interest and a
labelled probe.

Probes
• A probe is a short synthetic segment of DNA or
RNA, consisting of of 15 to 50 nucleotides
labeled with radioactive or non-radioactive
materials called a reporter (or a tracer).
• With radioactive reporters, the autoradiography
is used as detection system.
• Due to hazard of radioactive material, other types of
reporters were developed:
• Biotin reporter: the probe labeled with biotin,
then the enzyme-bound streptavidin is added,
then chromogenic substrate converted by the
enzyme into colored product which detected by
colorimetric assay.
 Probes
• Chemiluminescent molecule which
produce light when exposed to
excitor substances (UV), and the
signal detected by luminometer.
• Fluorescent molecules: probe
tagged with fluorescent molecule.

General workflow of hybridization

1. DNA denatured by alkali (NaOH) to separate double strands.
2. The single strand of DNA is then attached to a solid support such as
nitrocellulose or nylon membrane through its sugar phosphate
backbone and its bases project outward.
3. Next, a labelled probe is added to the membrane which hybridizes
with a DNA of interest if present.
4. Wash to remove unbound probes.
5. Detection of hybridization by detection system.
 Types of hybridization
• Hybridization can occur
• In solution with the participating
nucleic acid molecules being Incubation of membrane
present as single strands, with probe solution

• With one strand (target)

immobilized to a filter membrane
(solid phase), and the other strand
(probe) in solution, and
Membrane containing
• In situ, with the target strand fixed separated DNA fragments
in tissue sections, cells, or
chromosome smears, and the probe
present in solution.

Southern blot
• Southern blot is a method used for detection and quantification of a
specific DNA sequence in DNA samples.
• It one of the methods in which hybridization can be used.
• It has many applications like:
• Mutations and gene rearrangement studies (diagnosis of neonatal disease and
genetic disease).
• Paternity & maternity analysis, forensic studies, and personal identification.
 Southern blotting procedure
• DNA is digested with a restriction enzyme.
• Digested fragments are separated by gel electrophoresis.
• The double stranded DNA is denatured into single strands by
incubation with NaOH.
• The separated DNA fragments are transferred to a membrane
(blotting).
• The blot is incubated with a labelled probe, which base pairs with its
complementary DNA sequence if present (hybridization).
• Bound labelled probes are visualized (detection).
 Colony hybridization
• Colony hybridization is a method of
selecting bacterial colonies with
desired genes.
• Colonies are transferred to a
membrane, lysed, and DNA is
denatured by alkali treatment.
• Membrane is then incubated with
labeled DNA/RNA probe, which will
hybridize to target sequence.

Northern blot
• It is a traditional method that was used to
detect cellular mRNA and study gene
expression.
• The protocol is similar to Southern
blotting except that mRNA are not
digested with restriction enzymes
• mRNA is denatured to unfold into a
linear strand before it can bind
efficiently to nitrocellulose.
• formaldehyde and methyl mercuric
hydroxide can be used to denature the
mRNA.
• Alkali can not be used because they degrade
 In situ hybridization (ISH)
• In situ (inside the cell/tissue)
hybridization
• DNA probe for the detection of specific
sequences of nucleic acid in tissues.
• It can be used to detect mRNA in the cell
to find out whether a cell is expressing a
gene of interest in the cell.
• Fluorescent-tagged probes can used to
detect the presence of gene sequence on a
chromosome or a target mRNA in a cell.
• This technique is referred to as fluorescence
in situ hybridization (FISH).

Western blotting (immunoblotting)

• It is a technique for detecting specific proteins
separated by electrophoresis using labeled
antibodies.
• This is another blotting procedure but does not use
DNA/RNA probes and does not involve nucleic
acid hybridization.
 General workflow of WB
1. Cell lysate containing plenty of
proteins is obtained.
2. Proteins are separated using sodium
dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE).
• Proteins are all made negatively charged
with SDS treatment, so their separation is
based on differences in their sizes.

General workflow of WB
1. Separated proteins are transferred from
gel to a nitrocellulose or PVDF
(polyvinylidene difluoride) membrane
(blotting).
2. To prevent non-specific binding of
antibodies to the membrane, the membrane
is blocked by its incubation in a dilute
solution of protein such as 3% bovine
serum albumin (BSA) or non-fat dry milk.
Gel
Membrane
 General workflow of WB
1. The membrane blot is incubated in a
solution of primary antibody, which
binds specifically to the target protein.
2. After rinsing the membrane to remove
unbound primary antibody, the membrane
is exposed to another antibody known as
the secondary antibody.
1. The secondary antibody recognizes and
binds to the species-specific portion of the
primary antibody (e.g. anti-mouse if primary
is raised in mouse).
3. To allow detection of the target protein,
the secondary antibody is commonly

Comparison between different blotting techniques

Blot type Target Gel Probe Applications

mapping genomic
clones
Southern DNA DNA or RNA
Agarose estimating gene
numbers

RNA sizes and

Northern RNA DNA or RNA
Agarose expression

protein size and

Western Protein Antibodie
Polyacrylamide abundance