DNA hybridization

1. Molecularbiology&
Microbial genetics
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Molecular Hybridization
 Hybridization: the process of base-pairing between complementary ssDNA or RNA
from two different sources.
 Hybridization is the coming together, or binding, of two genetic sequences. The
binding occurs because of the hydrogen bonds between base pairs. Between ( A )
base and a (T )base, there are two hydrogen bonds; between a (C ) base and a (G )
base, there are three hydrogen bonds.
 Hybridization is also used in cloning and PCR. It can be used as an approach for
qualitative and quantitative analysis of DNA or RNA. Nucleic acid probes are used
extensively in many different diagnostic tests.
 When making use of hybridization in the laboratory, DNA must first be denatured,
usually by using heat or chemicals. Denaturing is a process by which the hydrogen
bonds of the original double-stranded DNA are broken, leaving a single strand of DNA
whose bases are available for hydrogen bonding.
Once the DNA has been denatured, a single-stranded radioactive probe [light
blue] can be used to see if the denatured DNA contains a sequence similar to that on
the probe. The denatured DNA is put into a plastic bag along with the probe and
some saline liquid; the bag is then shaken to allow sloshing. If the probe finds a fit, it
will bind to the DNA.
The fit of the probe to the DNA does not have to be exact. Sequences of varying
homology can stick to the DNA even if the fit is poor; the poorer the fit, the fewer the
hydrogen bonds between the probe [light blue] and the denatured DNA. The ability of
low-homology probes to still bind to DNA can be manipulated through varying the
temperature of the hybridization reaction environment, or by varying the amount of
salt in the sloshing mixture.

2. Molecularbiology&
Microbial genetics
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Probe
o Probe: a labeled, defined sequence used to search mixtures of nucleic acids for
molecules containing a complementary sequence
o Radioactive labeling: display and/or magnify the signals by radioactivity (32P)
o Non-radioactive labeling: display and/or magnify the signals by antigen labeling –
antibody binding – enzyme binding - substrate application (signal release) Such as
biotin, digoxigenin, fluorescent dye
o single-stranded , 25 to several thousand nucleotides in length
o Source of probe
– cDNA from highly expressed mRNA from a tissue
– homologous gene from a related organism
– DNA obtained from “reverse genetics” (protein-----DNA): If you have the
protein product of the gene in which you are interested….. sequence part of
the protein synthesize a short (>20 nucleotides) DNA probe based the protein
sequence using the genetic code use as your probe.
o Factors that affect hybridization characteristics
 Strand length
– The longer the probe the more stable the duplex
 Base Composition
– The % G:C base pairs are more stable than A:T
 Chemical environment
– The concentration of Na+ ions stablize
– Chemical denaturants (formamide or urea) destablize hydrogen bonds.
Autoradiography: A procedure in which a radioactive source is detected or
quantitated by its effect on a photographic film; a film is exposed to the radioactive
source, in the dark, and for an appropriate period of time, and is subsequently
processed. Autoradiography is used e.g. for investigating intracellular processes
(radioactive isotopes being incorporated into biomolecules) and also e.g. for detecting
bands of products (such as isotopically labeled fragments of DNA), in situ, after gel
electrophoresis. In general, optimal resolution may require the use of those isotopes
which have relatively low-energy emission (such as tritium, 3H) rather than those
(such as 32P) which have high energy emission. A sample can be exposed to X-ray
film and the exposed atoms turn black giving an image.
Techniques utilize molecular Hybridization
A. Transfer blotting (DNA or RNA is transferred and blotted from gel to membrane)
 Southern blotting
 Northern blotting
 Western blotting
B. Dot blotting & Slot blotting
C. In situ hybridization

3. Molecularbiology&
Microbial genetics
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Southern Blot
o The Southern blot is used to detect the presence of a particular bit of DNA in a
sample. The DNA detected can be a single gene, or it can be part of a larger piece of
DNA such as a viral genome.
o Major steps: electrophoresis --- transfer blotting ---- molecular hybridization
o probe suspect samples with single stranded, dye-labeled known DNA
o complete hybridization (exact sequence match) results in visible color
o can be performed on bacterial colonies or DNA samples separated on gel
electrophoresis by size (more specific)

4. Molecularbiology&
Microbial genetics
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Dot/Slot blots hybridization
♣ Dot blot: A simple probe-based method for detecting and/or quantitating a given
sequence of nucleotides in a sample. The samples separation is put directly onto the
membrane through dot /slot blot apparatus without electrophoresis.
A drop of the sample, on a membrane, is treated so that all the nucleic acid
molecules are released and bound to the membrane in single-stranded form. Target-
specific probes are then used to detect the given sequence; the probes are usually
labeled with e.g. digoxigenin or biotin. For quantitation of the target, the sample can be
diluted serially and each dilution probed in the way described; quantity is estimated by
comparing the strength of label from one or more dilutions of the sample with the
strength of label from control(s) of known concentration.
Slot blot is a similar method involving elongated (rather than dot-like) inoculations of
sample on the membrane.
Microarray (DNA chip)
• DNA microarray analysis follows the principles of Southern and Northern blot
analysis, but in reverse, with the sample in solution and the gene probes immobilized.
• DNA microarrays (DNA chips) are small, solid supports on to which DNA samples
corresponding to thousands of different genes are attached at known locations in a
regular pattern of rows and columns. The supports themselves may be made of glass,
plastic or nylon and are typically the size of a microscope slide.
• The DNA samples, which may be gene-specific synthetic oligonucleotides or cDNAs,
are spotted, printed, or actually synthesized directly on to the support. Thus each dot
on the array contains a DNA sequence that is unique to a given gene and which will
hybridize specifically to mRNA corresponding to that gene.
• Chip contains single stranded DNA probes (e.g. library of all viruses, library of all
E.coli strains, etc.)
• add patient sample to chip ,-binding of matching sequences causes color change
• colors detected by computer and scored for intensity,-read out indicates identity of
probe that bound best (identifies matching species)

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Microbial genetics
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• Consider an example of its use – to study the differences in the transcriptomes of a
normal and a diseased tissue. Total RNA is extracted from samples of the two tissues
and separately reverse transcribed to produce cDNA copies that precisely reflect the
two mRNA populations. One of the four dNTP substrates used for cDNA synthesis is
tagged with a fluorescent dye, a green dye for the normal cDNA and a red one for the
diseased-state cDNA. The two cDNA samples are then mixed together and hybridized
to the microarray (Fig. 1). The red and green-labeled cDNAs compete for binding to
the gene-specific probes on each dot of the microarray. When excited by a laser, a
dot will fluoresce red if it has bound more red than green cDNA; this would occur if
that particular gene is expressed more strongly (up-regulated) in the diseased tissue
compared with the normal one. Conversely, a spot will fluoresce green if that
particular gene is down-regulated in the diseased tissue compared with the normal
one.
Yellow fluorescence indicates that equal amounts of red and green cDNA have
bound to a spot and, therefore, that the level of expression of that gene is the same in
both tissues. A fluorescence detector comprising a microscope and camera produces
a digital image of the microarray from which a computer estimates the red to green
fluorescence ratio of each spot and, from this, the precise degree of difference in the
expression of each gene between the normal and diseased states. In order to ensure
the validity of the results, adequate numbers of replicates and controls are used and a
complex statistical analysis of the data is required.
• Since a full human genome microarray could have as many as 30 000 spots, the
amount of data generated is enormous. However, by clustering together genes that
respond in a similar way to a particular condition (disease, stress, drug etc.),
physiologically meaningful patterns can be observed.
• In terms of pure research, the clustering of genes of unknown function with those
involved in known metabolic pathways can help define functions for such orphan
genes.
• In theory, this type of array could be expanded to cover hundreds of known disease-
associated genes and so provide a global disease susceptibility fingerprint for an
individual.

6. Molecularbiology&
Microbial genetics
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ISH In situ hybridization
♣ ISH In situ hybridization: a probe-based procedure for detecting a target sequence
in situ (at a normal or aberrant location within cells or tissues or e.g. within an in vitro
preparation of chromosomes). A probe used for ISH – or a conjugate which is
subsequently bound to a hybridized probe – may exhibit any of various types of label.
Radioactive labels were widely used in the early protocols; fluorescent labeling (see
fish) is currently popular.
ISH has various applications in the study of structure and function in the biological
sciences. In medicine it can be used e.g. to detect certain pathogens in clinical
samples by using probes that bind to pathogen-specific sequences; in certain cases
(e.g. Mycobacterium tuberculosis) a pathogen may be detected by ISH significantly
earlier than it could be detected by culture. (ISH may also be able to distinguish
virulent from non-virulent strains of an organism by the use of probes for specific
virulence factors.)
♣ FISH Fluorescence in situ hybridization: a particular form of in situ hybridization
(ish). A hybridization technique that uses fluorochromes of different colors to enable
two or more genes to be located within a chromosome preparation in a single in situ
experiment.
FISH is precise because it uses DNA probes that are complementary to specific DNA
sequences. The probes are attached to molecules that fluoresce when illuminated,
producing a flash of color precisely where the probe binds to a chromosome in a
patient’s sample.
FISH can “paint” entire karyotypes by probing each chromosome with several
different fluorescent molecules. A computer integrates the images and creates a
unique false color for each chromosome. FISH reveals the extra chromosome 21 in
cells from a fetus with trisomy 21 Down syndrome.