GPB 501 Principles of Genetics
NUCLEIC ACID
HYBRIDIZATION
Presented by,
MARY NAVYA A S
2023508023
INTRODUCTION
The development of recombinant DNA techniques has spawned many
new approaches to the analysis of genes and gene products .
Geneticists can isolate and characterize essentially any gene from any
organism.
Once a gene has been cloned its expression can be investigated in even the
most complex organism such as humans.
Let’s consider some of the most important methods used to investigate the
structure of genes(DNA),their transcripts (RNA) and their final products (
usually proteins)
NUCLEIC ACID HYBRIDIZATION
Nucleic acid hybridization is a basic technique in molecular biology which
takes advantage of the ability of individual single standard nucliec acid
molecules to form double stranded molecules
Nucleic acid hybridization is a process used to identify specific DNA
sequences.
Specific DNA probes are denatured and annealed to sample DNA that has
also been denatured
Probe – a single-stranded nucleic acid that has been radiolabelled and is
used to identify a complimentary nucleic acid sequence that is membrane
bound
Probes used in hybridization reactions are usually chemically synthesized
DNA or RNA that has been labelled with a fluorescent dye or radioactive
isotope such as 32P
INSITU HYBRIDIZATION
Method of localizing, either mRNA within cytoplasm or DNA within the
chromosomes, by hybridizing the sequence of interest to a complimentary
strand of a nucleotide probe.
Radioactive copies of sat-DNA or its complementary RNA are prepared,
and used as probe.
Chromosomes in squash preparations are pretreated to expose and
denature their DNA
Squash preparation is then loaded with radioactive single-stranded probe.
Washed to remove nonhybridized radioactive probe and the location of
radioactivity in the Chromosomes is determined through autoradiography.
TYPES OF INSITU
HYBRIDIZATION
There are two basic ways to visualize RNA and DNA targets in situ-
Fluorescence(FISH) – Enables us to assay multiple targets simultaneously
and visualize co-localization within a single specimen
Chromogenic (CISH)- Enables to gain genetic information in the context of
tissue morphology.
Both use a labelled, target-specific probe that is hybridized with the sample,
the instrumentation used to visualize the samples is different for each
method.
Multiplex RNA visualization in cells using
ViewRNA FISH assays
SOUTHERN HYBRIDIZATION
The name of this technique is derived from the name of its inventor – E M Southern
DNA : DNA hybridization forms it’s basis
The following steps describe the Southern transfer procedure.
Digest DNA with the restriction enzyme of choice.
Load the digestion onto a agarose gel and apply an electrical current. DNA is negatively
charged so it migrates toward the “+” pole. The distance a specific fragment migrates is
inversely proportional to the fragment size.
Stain the gel with EtBr, a fluorescent dye which intercalates into the DNA molecule. The
DNA can be visualized with a UV light source to assess the completeness of the digestion.
Denature the double-stranded fragments by soaking the gel in alkali (>0.4 M NaOH)
Transfer the DNA to a filter
membrane (nylon or nitrocellulose)
by capillary action. This process is
known as [Link] a
Southern transferst setup contains
(from bottom to top):
buffer
sponge
filter paper
the gel containing the nucleic acid
a nylon or nitrocellulose
membrane
filter paper
paper towels to catch the buffer
that passed through all of the
above
Nitrocellulose membrane is now removed from blotting [Link] is
permanently immobilized on membrane by baking it at 80°C in vacuo.
Prepare a probe by nick translation or random, oligo-primed labelling.
Add the probe to a filter (nylon or nitrocellulose) to which single-stranded
nucleic acids are bound. (The filter is protected with a prehybridization
solution which contains molecules which fill in the spots on the filter
where the nucleic acid has not bound.
Hybridize the single-stranded probe to the filter-bound nucleic acid for 24
hr. The probe will bind to complementary sequences.
Wash the filter to remove non-specifically bound probe.
Expose the filter and determine whether binding occured
NORTHERN HYBRIDIZATION
Developed by James Alwine,George stark and David Kemp(1977)
In this technique RNA bands are separated by gel electrophoresis and the RNA
bands are transferred onto a suitable membrane eg:
diazobenzyloxymethyl(DBM)paper or nylon membrane
The bands are hybridized with radioactive single stranded DNA probe and the
bands showing hybridization are detected by autoradiography.
extension of Southern blotting technique
DIFFERENCE BETWEEN SOUTHERN
AND NORTHERN HYBRIDIZATION
In Southern hybridization DNA is separated by gel electrophoresis,while
in Northern blotting RNAs are separated.
In Southern hybridization DNA has to be denatured before blotting while
the step is not needed in Northern hybridization
Nitro cellulose membrane is generally not used for Northern while it is
often used for southern
Finally hybridization with the probe produces DNA- DNA hybrid
molecules but RNA -DNA molecules in Northern hybridization.
WESTERN BLOTTING
Proteins are separated by
polyacrylamide gel
electrophoresis.
Protein bands are
transferred onto a
nitrocellulose or nylon
membrane
Initially this was achieved by
capillary movement of buffer
(Capillary blotting)but now-
a-days by
electrophoresis(Electrophore
tic blotting)
CONTD..
Specific protein bands are identified in a variety of ways
1) Antibodies are most commonly used as probes for detecting specific
antigens
2) Lectins are used as probes for identification of glycoproteins.
Identification process is often based on ‘Sandwich reaction’
In this, a species-specific second antibody or protein A of Staphylococcus
aureus
or streptavidin is used to bind to the antibodies bound to the protein bands.
Second molecules may be labelled with radioactive,enzyme or fluorescent
tags.
DOT BLOT TECHNIQUE
For detecting the presence of sequence being transferred in a number of
suspected transgenic individuals, the presence of specific mRNA in several
such individuals.
Procedure
[Link] DNA or RNA from each of several different individuals or tissues
is transferred onto a nitrocellulose filter in form of a dot .Several samples
dot-blotted onto a single filter
[Link] is first denatured and then filter is baked at 80°C to fix DNA firmly
onto filter
Filter is pretreated to prevent nonspecific binding of the probe to the filter
[Link] is then treated with radioactive single-stranded DNA probe under
conditions favouring hybridization. Filter is then washed repeatedly to
remove free Probes
[Link] dots having
concerned DNA or RNA
sequence will hybridize
with radioactive probe.
Dots are detected by
autoradiography.
Dots that show up in the
autoradiograph denote
individuals or tissues in
which DNA or RNA
sequence corresponding to
the probe is represented.
COLONY HYBRIDIZATION
To identify those bacterial colonial in a plate,which contain a specific DNA
sequence that was introduced into them through genetic engineering;the same
sequence is represented by the probe used in hybridization experiment.
Procedure
[Link] cells subjected to transformation are plated on to a suitable agar plate;
this is the master plate.
[Link] colonies of master plate are replica plated onto a nitrocellulose filter
membrane placed on agar medium.
For replica plating, a block of wood or cork, suitable for master plate, is covered
with velvet cloth.
Block is sterilized and then lowered into master plate till velvet touched all
colonies
CONTD..
The block is withdrawn and gently lowered onto nitrocellulose filter so
that bacterial colonies sticking onto velvet are transferred onto the filter.A
reference point is marked both on master plate and on replica plate to
facilitate later comparisons
[Link] colonies appear on the filter, the filter is removed from agar plate
and treated with alkali to lyse the bacterial [Link] also denatures DNA
of these cells.
[Link] filter is treated with proteinase K to digest and remove the
[Link] denatured DNA remains bound to the filter.
[Link] filter is now baked at 80°C to fix the DNA onto filter; this yields
DNA print of bacterial colonies in the same relative positions as in the
master plate.
APPLICATIONS
DNA and RNA Detection: Used for identifying specific DNA or RNA
sequences in a sample, aiding in diagnostics and research.
Gene Expression Studies: To assess the activity of genes by detecting
messenger RNA levels
Comparative Genomic Analysis: Facilitates comparison of nucleic acid
sequences among different organisms, shedding light on evolutionary
relationships.
Molecular Cloning: Essential in creating recombinant DNA molecules by
hybridizing target DNA with vector DNA, a crucial step in gene cloning.
In Situ Hybridization: Localizes specific nucleic acid
sequences within cells or tissues, allowing researchers to
study gene expression patterns in their native
environments.
Diagnostic Applications: Applied in medical diagnostics
for detecting pathogens, mutations, or genetic variations
associated with diseases.
Forensic Analysis: Utilized in DNA fingerprinting for
identifying individuals, solving crimes, and establishing
paternity.
LIMITATIONS
Specificity: Hybridization may not be entirely specific, leading to potential
cross-reactivity with similar sequences
Sensitivity: Detection sensitivity can be affected by factors like probe
concentration and target abundance,
Denaturation Issues: Ensuring complete denaturation of nucleic acid samples
can be challenging, affecting the accuracy of hybridization.
.Sequence Variability: Hybridization may be less effective in regions with
high sequence variability or secondary structures.
Probe Design Challenges: Designing specific and effective probes for
hybridization can be challenging, especially for complex genomes.
Temperature Sensitivity: Hybridization is temperature-sensitive, and
variations can impact the stability of the formed hybrids.
REFERENCE
Genetics B D Singh
[Link] Nucleic acid hybridization & Expression analysis
[Link] Hybridization
[Link] Nucleic acid hybridization an overview
