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‡ has emerged form the development in the
area of genetic engineering and molecular
biology.
‡ this technique is used to detect the presence
of DNA from pathogens in clinical specimens
and locate specific genes in cell.
‡ is a method for identifying closely related
nucleic acid molecules within two
populations, a complex target population and
a comparatively homogeneous probe
population
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Definition of nucleic acid hybridization

‡ Nucleic acid hybridization involves mixing

single strands of two sources of nucleic acids,
a probe which typically consists of a
homogeneous population of identified
molecules (e.g. cloned DNA or chemically
synthesized oligonucleotides) and
a target which typically consists of a complex,
heterogeneous population of nucleic acid
molecules.
‡ If either the probe or the target is initially
double-stranded, the individual strands must be
separated, generally by heating or by alkaline
treatment. After mixing single strands of probe
with single strands of target, strands with
complementary base sequences can be allowed
to reassociate. Complementary probe strands
can reanneal to form homoduplexes, as can
complementary target DNA strands.
‡ However, it is the annealing of a probe DNA
strand and a complementary target DNA strand
to form a labeled probe-target heteroduplex that
defines the usefulness of a nucleic
acid hybridization assay. The rationale of
the hybridization assay is to use the
identified probe to query the target DNA by
identifying fragments in the complex target
which may be related in sequence to the probe.
 Úm
 

h. DNA from a virus or cell is denatured with alkali
to separate the strands.
2. The single strands of DNA are then attached to
a solid support such as nitrocellulose or nylon
membrane so that the strands do not reanneal.
3. The DNA is attached to the membrane by its
sugar-phosphate backbone with the nitrogenous
bases projecting outward.
4. To characterized or identify the target DNA, a
single-stranded DNA or RNA molecule of
known origin called a probe is added to the
membrane in a buffered solution.
 
 

‡ this allows the formation of hydrogen bonds
between complementary bases.
‡ the probe is so called because if it used to
seek or probe for DNA sequences, is
labelled with a reporter group, which may be
a radioactive atom or an enzyme whose
presence can be easily detected.
‡ the probe is allowed to react with the target
DNA; then any unreacted probe is removed
by washing in buffered solutions.



‡ after the washes, all that remains on the

nitrocellulose is the target DNA and any
probe molecules that have attached to
complementary sequences in the target
DNA, forming stable hybrids.
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V. Hybridization of target and probe DNAs is
¶detected by assaying for the probes reporter
group.
6. If the reporter group is detected, hybridization
has taken place. If no reporter group is detected,
it can be assumed that the target molecule does
not have sequences that are complementary to
those of the probe and hence sequences that are
complementary to those of the probe and hence
the gene or DNA segment sought is not present
in the sample.
 r
'our components of DA
Hybridization

h. Target DNA
2. Probe
3. Detection System
4. 'ormat
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h. Dot Blot Assay - a specified volume of
sample or specimen is spotted onto a small
area of nitocellulose membrane.
2. Southern Hybridization Assays ² invlove
restriction enzyme digestion and agarose
gel electrophoresis of the target DNA prior
to the hybridization assay.
- the different bands on the agarose gel are
transferred by capillary action onto a nitocellulose
or nylon membrane in a blot apparatus.
Dot blot hybridization analysis of DNA from the LMW and HMW
gel fractions with probes for repetitive sequences known to be present
in T.brucei: subtelomeric repeats (subtel), the conserved second part
of VSG genes (VSG), 70, h77 and V0 bp and rDNA repeats and
kDNA minicircles; hybridization signals are quantitated relative to
non-fractionated control DNA (bloodstream form DNA digest;
tubulin is h).
Southern Hybridization Assays
In Situ Hybridization Assays
- during the transfer, each if the DNA bands is
transferred onto the membrane in the same
relative position that it had in the gel.
3. In Situ Hybridization Assays ² involve
the probing of intact cells or tissue
sections affix to a microscope slides.
- this type of solid phase assay has the advantage
that one cannot only detect the presence of
target DNA in intact cells but also determine
the location of such target DNA within a tissue.
- an important application of in situ
hybridization is for the detection of viruses and
certain types of bacteria within infected cell.
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