Subject Name: Genetic Engineering-II

Subject Code: MTI-605

Unit No:5
Unit Name: Molecular Diagnostics
Faculty Name : Dr. V. Suresh Gupta

Sub Topic Name Hybridization

Hybridization

Hybridization
Nucleic Acid Diagnostic Systems
Genetic material of organism contains essential informn that contributes to its various features &
characteristics.
- For ex., bacterial pathogenicity due to presence of specific or set of genes.
Similarly, alteration of a gene may cause inherited genetic disease in humans.
In theory, nucleotides seq contributing to particular biological characteristic - distinctive signature
that, if detectable, can be used as a definitive diagnostic determinant.
Nucleic acid hybridization is the basis for rapid & reliable assays.
The physical basis of these systems is precise nucleotide base pairing & hydrogen bonding between
one string of nucleotides and a complementary nucleotide sequence.
A general laboratory nucleic acid hybridization scheme is as follows:
1. Bind single-stranded DNA (the target) to a membrane support.
2. Add single-stranded labeled DNA (the probe) under appropriate conditions of temperature & ionic strength to
promote base pairing between the probe and the target DNAs.
3. Wash the support to remove excess unbound labeled probe DNA.
4. Detect the hybrid seqs that form between the probe & target DNA.

A nucleic acid hybridization diagnostic test has 3 critical elements: probe DNA, target DNA, & signal
detection.
This type of detection system can be both extremely specific & highly sensitive.

Hybridization

What is hybridization?
Hybridization is the process of combining
two complementary single-stranded DNA
or RNA molecules and allowing them to
form a single double-stranded molecule
through base pairing.
DNADNA hybridization generally refers
to a molecular biology technique that
measures the degree of genetic similarity
between pools of DNA sequences
It is usually used to determine
the genetic distance between two
organisms.
This has been used a lot in phylogeny and
taxonomy.
5

Hybridization
Hybridization Probes
To be effective, a nucleic acid hybridization probe must have a high degree of
specificity.
In other words, probe must hybridize exclusively to the selected target nucleic acid
sequence.
False positives (i.e., responses in the absence of the target sequence) & false negatives
(i.e., no response when the target is present) severely undermine utility of a diagnostic
procedure.
Probes can be specific at different organismic levels. They can distinguish between 2
or more species, determine particular strains within a given species, or identify
differences between genes.
Depending on the requirements of the test protocol, probes can be DNA or RNA,
long (>100 nucleotides) or short (<50 nucleotides), and chemically synthesized, cloned
intact genes, or isolated regions of a gene.
Sequences that might make effective probes can be isolated in a number of ways.
For ex., DNA from pathogenic organism can be cut with a restriction endonuclease
and cloned into a plasmid vector.
6

Hybridization
Recombinant plasmids are screened with the genomic DNA from both pathogenic &
nonpathogenic strains.
Those plasmids that contain sequences that hybridize only to the pathogenic strain form
the basis for species-specific, and even strain-specific, probes (Fig. 9.10).
Additional hybridization tests with DNA from a wide range of organisms are then conducted
to ensure that the candidate probe sequences do not cross- hybridize.
Each potential probe is also tested under simulated sample conditions, including presence
of mixed cultures, to determine its level of sensitivity.
It is important to note that knowledge of the genomic sequence of a large number of
bacterial pathogens (currently several hundred) has facilitated identification of unique
stretches of DNA that could be used as probes.
The ability to perform nucleic acid probe diagnostic assays directly on available samples
without either additional culturing or time-consuming extraction procedures is extremely
desirable, especially with clinical specimens.
Researchers have successfully used probes that hybridize to target DNA from fecal samples,
urine, blood, throat washes, and tissue samples without extensive DNA purification.
If a target sequence is rare in the working sample, PCR can be used to amplify it.

Hybridization

Hybridization

Nucleic acid blot hybridization

Nucleic acid hybridization is usually performed with target DNA that has been
immobilized onto membranes (from Southern blots, by spot loading DNA onto
membranes or after lysis of the bacterial colonies on a replica filter) and a probe
in a liquid phase.
Standard nucleic acid hybridization assays involve using a labeled nucleic acid
probe to identify related DNA or RNA molecules (that is, ones with a significantly
high degree of sequence similarity) within a complex mixture of unlabeled nucleic
acid molecules, the target nucleic acid.

Hybridization
1. Diagnosis of Malaria
Diagnostic protocol utilizing DNA probe developed for detection of Plasmodium
falciparum.
Malaria, affects ~1/3rd of world's population - infects & destroys RBCs, leading
to fever and, in severe cases, damage to brain, kidneys & other organs.
Sensitive, simple & inexpensive methods reqd to identify source(s) of parasite in
various localities, to assess progress of eradication programs, and to facilitate
early treatment.
Currently, microscopic examination of blood smears or immunological detection
of parasite Ags, effective but labor-intensive & time-consuming processes,
especially given large nos of samples to be examined. [ELISAs - rapid & amenable
to automation - do not always discriminate current & past infections because designed
simply to detect anti-Plasmodium Abs in blood].

10

Hybridization
DNA diagnostic procedure selectively measures only current infections, i.e.,
presence of DNA-containing organisms, was developed by using highly
repeated DNA from P. falciparum.
First, a genomic library of parasite DNA screened with labeled wholegenome parasite DNA.
The most intensely labeled hybridizing colonies were selected because they
expected to contain repetitive DNA.
DNA from each of the selected colonies was then tested for its ability to
hybridize with DNA from several other Plasmodium species that do not cause
malaria.
DNA sequence that was chosen as a specific probe hybridized with P.
falciparum but not with Plasmodium vivax, Plasmodium cynomolgi, or human
DNA, despite the fact that P. vivax causes a less severe form of malaria.

11

Hybridization
This probe can detect as little as 10 picograms of purified P. falciparum DNA (or) 1
nanogram (ng) of P. falciparum DNA in blood.
More than 100 different DNA diagnostic probes isolated & characterized for detection
of various pathogenic strains of bacteria, viruses & parasites.
-For ex., probes developed for the diagnosis of human bacterial infections caused by
Legionella pneumophila (respiratory failure), Salmonella enterica serovar Typhi (food
poisoning), Campylobacter hyointestinalis (gastritis), and enterotoxigenic E. coli
(gastroenteritis).
Clearly, this is just the "tip of the iceberg," because in principle, nearly all pathogenic
organisms can be detected by this procedure.

12

Hybridization
2. Detection of T. cruzi
Protozoan parasite Trypanosoma cruzi - causative agent of Chagas disease [invade
liver, spleen, lymph nodes & central nervous system - where they multiply & destroy
infected cells.
T. cruzi - quite common in Latin America, spread by insects, responsible for ~50,000
deaths / yr.
Diagnosis of acute Chagas disease - microscopic examination of a fresh blood
sample.
Alternatively, a test that takes longer time but ensures that parasite not overlooked
entails feeding patient's blood to uninfected insects and then microscopic examining
of insects' intestines for parasites about 30 - 40 days later.
Both of these tests are laborious, time-consuming, and costly.
Chagas disease also diagnosed by immunological tests; however, produce false+ve
responses.
At present, PCR assays for Chagas disease used as adjuncts to traditional diagnostic
procedures.

13

Hybridization

In one PCR-based assay procedure - 188 bp DNA fragment

present in multiple copies in T. cruzi genome but absent from
genomic DNA of several related parasites is the target sequence.
The presence of amplified 188-bp DNA fragment is readily
detected by PAGE.
- In general, with minor variations in methodology, such as primer
seqs, PCR can facilitate detection of a wide range of bacteria,
viruses, & parasites.
- Currently, several PCR-based diagnostic kits been approved for
use by U.S. FDA for the detection & quantitation of HIV,
Mycobacterium tuberculosis & C. trachomatis.

14

Hybridization
Nonradioactive Hybridization Procedures
In many research laboratories, nucleic acid hybridization is routinely detected by
labeling probe with a radioactive isotope, commonly phosphorus-32. High specific
activity ensures an excellent signal-to-noise ratio.
In a standard detection system, a radiolabeled probe is mixed with target DNA
that is bound to a membrane support. After the support is washed free of
nonhybridized probe DNA, the presence of radioactivity is determined by laying
membrane on X-ray film (autoradiography).
However, phosphorus-32 - short-lived, potentially dangerous & requires special
laboratory equipment for handling & safe disposal, so nonradioactive systems for
indicating hybrid DNA formation developed.
Nonradioactive detection systems achieve signal amplification by enzymatic
conversion of either chromogenic or chemiluminescent substrates [Chromogenic
substrates change color & chemiluminescent substrates give off light when they
are converted into a specific product by an appropriate enzyme].

15

Hybridization
The signal is detected, in most of these systems, by incorporating biotinlabeled nucleotides into the DNA probe and following a more or less standard
procedure:
1. Biotin-labeled probe is hybridized to the target DNA (Fig. 9.11 A).
2. Either avidin (chicken egg white protein) or streptavidin (bacterial analogue
of avidin), is added (Fig. 9.11B).
3. A biotin-labeled enzyme, such as alkaline phosphatase (or) peroxidase, is
added (Fig. 9.11C).
4. Depending on which biotin-labeled enzyme was used in the previous step,
either a chromogenic (or) a chemiluminescent substrate is added, and either
the color change (or) the light produced as a consequence of the conversion
of substrate into product is measured (Fig. 9.11D).

16

Hybridization

17

Hybridization
Alternatively, following hybridization with a biotin-labeled probe in step 2
above, a streptavidin-enzyme complex with an available biotin- binding site
can be added.
Both avidin & streptavidin bind very tightly (Kd [dissociation constant] = ~10 15)
to biotin; in addition, each of these proteins has 4 separate biotin-binding
sites, so a single molecule of avidin or streptavidin can bind both a biotinlabeled enzyme & a biotin-labeled probe. Enzymatic activity is not impaired by
biotin labeling or binding to streptavidin.
In chromogenic detection systems - enzyme action on substrate creates a
colored insoluble dye that remains at the site of hybrid DNA.
In chemiluminescent systems - enzymatic alteration of substrate generates a
product that emits light at the site of the hybrid DNA.

18

Hybridization
Nonradioactive systems have other advantages:
1. biotin-labeled DNA is stable for at least 1 year at room temperature,
2. devices that detect chemiluminescence are as sensitive as those that
detect radioactive signals, and
3. detection of the emitted light with either X-ray film or a luminometer, or
scoring of a color change, can be completed within a few hours.
Use of chemiluminescence, which is more sensitive than chromogenic
dyes, is becoming the detection signal system of choice for many nucleic
acid probe- based diagnostic assays.
For PCR-based assays, amplification product can be labeled by a
fluorescent dye that is bound to the 5' end of each primer.
A fluorescent compd emits light of a longer wavelength after it absorbs
light of a shorter wavelength.

19

Hybridization
Fluorescein, which appears green
under certain wavelengths of light, &
rhodamine, which appears red, are
often used for this purpose.
After PCR amplification of a target DNA
with fluorescence-labeled primers, the
primers are separated from the
amplification product and the presence
of the label is detected (Fig. 9.12).
If the target DNA is not present in the
sample, then no fluorescent product
will be observed.
This system is not only sensitive, it is
also quite rapid, since it is not
necessary to run a gel to separate the
amplified target DNA.
20

Hybridization

DIFFERENT TYPES OF
HYBRIDIZATION TECHNIQUES
SOUTHERN BLOTTING
NORTHEN BLOTTING
DOT BLOTTING
WESTERN BLOTTING
PLAQUE OR COLONY BLOTTING

21

Hybridization

SOUTHERN HYBRIDIZATION
A Southern blot is a method used in molecular biology for detection of a
specific DNA seq in DNA samples.
Southern blotting combines transfer of electrophoresis-separated DNA
fragments to a filter membrane & subsequent fragment detection by
probe hybridization.
Southern blots performed with restriction enzyme-digested genomic
DNA may be used to determine the no. of sequences (e.g., gene
copies) in a genome.
A probe that hybridizes only to a single DNA segment that has not been
cut by the restriction enzyme will produce a single band on a Southern
blot, whereas multiple bands will likely be observed when the probe
hybridizes to several highly similar sequences (e.g., those that may be
the result of sequence duplication).
22

Hybridization

23

Hybridization

Southern Hybridization - Applications

To locate the exact position of a cloned gene within a recombinant DNA
molecule. Large DNA fragment, small gene size. Southern blots of cloned
genomic DNA fragments can be probed with cDNA molecules to find
which parts of the genomic clone correspond to the cDNA fragment. If
the southern blot contains genomic DNA fragments from the whole
genome, the probe will give information about the size of the fragment
the gene is on the genome and how many copies of the gene are present
in the genome.
Used in the estimation of copy number of a specific gene. several lanes
with different copy numbers of the gene to which you are probing are run
and the hybridization intensities are compared with a companion
genomic southern experiment. This is called a reconstruction experiment.
To determine if a phenotypic mutation is due to a structural change in
the gene controlling the trait of interest. Hybridization pattern changes
with indels.
24

Hybridization

Southern Blot

25

DNA (genomic or other source) is digested with a restriction enzyme and separated
by gel electrophoresis, usually an agarose gel.
Because there are so many different restriction fragments on the gel, it usually
appears as a smear rather than discrete bands. The DNA is denature into single
strands by incubation with NaOH.
The DNA is transferred to a membrane which is a sheet of special blotting paper.
The DNA fragments retain the same pattern of separation they had on the gel.
The blot is incubated with many copies of a probe which is single-stranded DNA.
This probe will form base pairs with its complementary DNA sequence and bind to
form a double-stranded DNA molecule. The probe cannot be seen but it is either
radioactive or has an enzyme bound to it (e.g. alkaline phosphatase or horseradish
peroxidase).
The location of the probe is revealed by incubating it with a colorless substrate that
the attached enzyme converts to a colored product that can be seen or gives off light
which will expose X-ray film. If the probe was labeled with radioactivity, it can
expose X-ray film directly.

Hybridization

Southern Blot Method

26

Hybridization

NORTHERN HYBRIDIZATION
This technique is for the specific
identification of RNA, It is an extension
method of southern blotting
Northern blotting involves the use of
electrophoresis to separate RNA samples
by size and detection with a hybridization
probe complementary to part of or the
entire target sequence.
With northern blotting it is possible to
observe cellular control over structure
and function by determining the particular
gene
expression
levels
during
differentiation, morphogenesis, as well as
abnormal or diseased conditions.

27

Hybridization

Northern Hybridization - Applications

the detection of RNA size, the observation of alternate splice products, the use of probes
with partial homology, the quality and quantity of RNA can be measured on the gel prior to
blotting, and the membranes can be stored and reprobed for years after blotting.
allows in observing a particular gene's expression pattern between tissues, organs,
developmental stages, environmental stress levels, pathogen infection.
used to show over expression of oncogenes and down regulation of tumor-suppressor
genes in cancerous cells when compared to 'normal' tissue.
To detect gene expression in the rejection of transplanted organs.
useful to determine whether a cDNA clone used as a probe is full-length or whether it is one
of a family of related transcripts.
Northern blots can help to identify whether a genomic clone has regions that are
transcribed and, if the RNA on the blot is made from different tissues, where these
transcripts are made.
With northern blotting it is possible to observe cellular control over structure and function
by determining the particular gene expression levels during differentiation, morphogenesis,
as well as abnormal or diseased conditions.
28

Hybridization

Northern Blot
RNA (either total RNA or just mRNA) is separated by gel electrophoresis,
usually an agarose gel. Because there are so many different RNA molecules on
the gel, it usually appears as a smear rather than discrete bands.
The RNA is transferred to a sheet of special blotting paper called
nitrocellulose, though other types of paper, or membranes, can be used. The
RNA molecules retain the same pattern of separation they had on the gel.
The blot is incubated with a probe which is single-stranded DNA. This probe
will form base pairs with its complementary RNA sequence and bind to form a
double-stranded RNA-DNA molecule. The probe cannot be seen but it is
either radioactive or has an enzyme bound to it (e.g. alkaline phosphatase or
horseradish peroxidase).
The location of the probe is revealed by incubating it with a colorless
substrate that the attached enzyme converts to a colored product that can be
seen or gives off light which will expose X-ray film. If the probe was labeled
with radioactivity, it can expose X-ray film directly.
29

Hybridization

30

Hybridization

WESTERN BLOTTING
This involves the identification of protein. Useful to understand nucleic acid function
particularly during the course of gene manipulation
The western blot (sometimes called the protein immunoblot) is a widely used analytical
technique used to detect specific proteins in a sample of tissue homogenate or extract.
It uses gel electrophoresis to separate native proteins by 3-D structure or denatured
proteins by the length of the polypeptide. The proteins are then transferred to a
membrane (typically nitrocellulose or PVDF), where they are stained with antibodies
specific to the target protein.
The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a
human serum sample. Proteins from known HIV-infected cells are separated and blotted
on a membrane as above. Then, the serum to be tested is applied in the primary antibody
incubation step; free antibody is washed away, and a secondary anti-human antibody
linked to an enzyme signal is added. The stained bands then indicate the proteins to
which the patient's serum contains antibody.

31

Hybridization

32

Hybridization

33

Hybridization

34

Hybridization

35

Hybridization

Western Blotting Applications

Western blotting can be used to identify a specific antibody
in a mixture.
in confirmatory testing for HIV, where Western blotting is
used to determine whether the patient has antibodies that
react with one or more viral proteins.
used as the definitive test for mad cow disease.
used as a confirmatory test for Hepatitis B infection

36

Hybridization

DOT BLOTTING
Modified southern & northern blotting technique.
It is a technique in molecular biology used to detect biomolecules, and for detecting,
analyzing, and identifying proteins.
In a dot blot the biomolecules to be detected are not first separated by electrophoresis.
Instead, a mixture containing the molecule to be detected is applied directly on a
membrane as a dot, and then is spotted through circular templates directly onto the
membrane or paper substrate.
This differs from the western blot because protein samples are not separated
electrophoretically. This is then followed by detection by either nucleotide probes (for
a northern blot and southern blot) or antibodies (for a western blot).
The technique offers significant savings in time, as chromatography or gel
electrophoresis, and the complex blotting procedures for the gel are not required.
The sensitive dot blot test can be used to detect the Chlamydia trachomatis infection
and other sexually transmitted diseases. Dot blot is used to detect Antidiacyltrehalose
Antibodies in Tuberculous patients &Typhoid Fever.
37

Hybridization

NUCLEIC ACID BLOT HYBRIDIZATION

Nucleic acid hybridization is usually performed with target DNA that has
been immobilized onto membranes (from Southern blots, by spot loading
DNA onto membranes or after lysis of the bacterial colonies on a replica
filter) and a probe in a liquid phase.
Standard nucleic acid hybridization assays involve using a labeled nucleic
acid probe to identify related DNA or RNA molecules (that is, ones with a
significantly high degree of sequence similarity) within a complex mixture of
unlabeled nucleic acid molecules, the target nucleic acid.

38

Hybridization

PLAQUE OR COLONY BLOTTING

Plaque blotting is similar to colony blotting, the only difference is that instead of bacterial
colony, a plaque is transferred onto the membrane
The greatest advantage of this method is that several identical DNA prints can be easily
made from a single master plate containing bacteria/plaques which are to be made
Here the DNA is fixed tightly to membrane by either UV cross linking or oven baking.
In this procedure, the bacterial colonies to be screened are transferred onto nitrocellulose
or nylon membrane by using replica plating.

39

Hybridization
What is reverse hybridization?
Reverse hybridization assays offer a platform for highly specific probe hybridization.
Specific DNA probes are immobilized on a solid carrier, such as nitrocellulose strips (or) Luminex
beads.
The test procedure comprises 3 parts:
Isolation of the DNA from the sample
Amplification of target DNA using PCR
Detection of the biotinylated product
DNA or RNA derived amplification products can be denatured and hybridized to the immobilized
probes. After stringent washing steps, the specific hybrids can be detected. On reverse hybridization
strips, this results in a visible hybridization pattern.
In the Luminex assay format, this can be measured by a specific Luminex reader.
Reverse hybridization assays have the following advantages:
Hybridization is highly specific, allowing single nucleotide mismatch detection
Detection is very sensitive, especially to detect minority species amplimers e.g. in mixed infections
The read-out can be performed manually or automated
The test is fast (~8 hours, including amplification)
40

Hybridization
Ref:
Molecular Diagnostics;
Chap 9, Glick and Pasternak P-333-378.

41

Thank You