IN-SITU HYBRIDIZATION

&
BIOMARKERS
[RFLP: RESTRICTION FRAGMENT
LENGTH POLYMORPHISM]
[RAPD: RANDOM AMPLIFIED
POLYMORPHIC DNA]
In situ hybridization is a procedure that allows locating the positions of specific
DNA sequences on chromosomes.

• The first in situ hybridization experiments was developed by Gall & Pardue
in 1969, many variations of the procedure have been developed, and its
sensitivity has increased enormously.

• Today, most in situ hybridization procedures use fluorescent probes to

detect DNA sequences, and this process is commonly referred to
as FISH (fluorescence in situ hybridization).

• A variety of FISH procedures are available and it is used to diagnose many

types of chromosomal abnormalities in patients.

• The success of FISH, and all other methods of in situ hybridization,

depends on the remarkable stability of the DNA double helix.

In Situ Hybridization (ISH)

 • It is a technique that allows for defined localization of a specific segment of
nucleic acid within a histologic section.

• The underlying basis of ISH is that nucleic acids, if preserved adequately

within a histologic specimen, can be detected through the application of a
complementary strand of nucleic acid to which a reporter molecule is
attached.

• Visualization of the reporter molecule allows to localize DNA or RNA

sequences in a heterogeneous cell populations including tissue samples and
environmental samples.

• Riboprobes also allow to localize and assess degree of gene expression. The
technique is particularly useful in neuroscience.

In situ hybridization probes

• Double-stranded DNA (dsDNA) probes

• Single-stranded DNA (ssDNA) probes

• RNA probes (riboprobes)

• Synthetic oligonucleotides (PNA, LNA)

Labeling techniques

• Radioactive isotopes

– 32
P

– 35
S

– 3
H

• Non-radioactive labels

– biotin

– digoxigenin
 – fluorescent dye (FISH)

Applications of In Situ Hybridization

• Microbiology (classic target - 16S rRNA) - morphology and population

structure of microorganisms

• Pathology (pathogen profiling, abnormal gene expression)

• Developmental biology (gene expression profiling in embryonic tissues)

• Karyotyping and phylogenetic analysis (unique FISH patterns on individual

chromosomes, chromosomal aberrations)

• Physical mapping (mapping clones on chromosomes and direct assignment

of mapped clones to chromosomal regions associated with
heterochromatin or euchromatin).

Fluorescence In-Situ Hybridization (FISH)

• Fluorescence in situ hybridization (FISH) is a molecular biology method used
to visualize and enumerate specific types of microorganisms or groups of
microorganisms in an environmental sample.

• The method does not require isolation or cultivation of microorganisms and

allows for examination of microorganisms in complex environmental
samples with minimal disruption of the natural microbial community. Since
its introduction in the late 1980’s, FISH has been used in medical and
developmental biology and environmental bacteriology.

• Today, FISH is considered to be a powerful tool for phylogenetic, ecological,

diagnostic, and environmental microbiology studies.

Procedure

The basic FISH procedure includes:

• fixation and permeabilization

• hybridization

• washing

• microscopy (for counting and visualization) or flow cytometry.

• FISH technique is dependent upon hybridizing a probe with a fluorescent

tag, complementary in sequence to a short section of DNA on a target
gene.

• The tag and probe applied to a sample of interest under condition that
allow for the probe to attach itself to the complementary sequence in the
specimen.

• After sample treatment, excess flurophore is washed away and the sample
can be visualized under a fluorescent microscope.

The basic elements of FISH are a DNA probe and a target sequence.

Before hybridization, the DNA probe is labeled by various means, such as

nick translation, random primed labeling, and PCR.
Two labeling strategies are commonly used: indirect labeling (left panel) and
direct labeling (right panel).
For indirect labeling, probes are labeled with modified nucleotides that
contain a hapten, whereas direct labeling uses nucleotides that have been
directly modified to contain a fluorophore.
 The labeled probe and the target DNA are denatured.

Combining the denatured probe and target allows the annealing of

complementary DNA sequences.

If the probe has been labeled indirectly, an extra step is required for
visualization of the non-fluorescent hapten that uses an enzymatic or
immunological detection system.
Whereas FISH is faster with directly labeled probes, indirect labeling offers the
advantage of signal amplification by using several layers of antibodies, and it
might therefore produce a signal that is brighter compared with background
levels.
FIGURE: STEPS INVOLVED IN FISH

Molecular Markers
• In recent years recombinant DNA technology and PCR technology have
helped in construction of genetic, cytogenetic and physical maps of genome
of plants and animals using molecular markers.

• The markers revealing variations at DNA level are referred to as the

molecular markers.
 • A large number of genetic polymorphism occuring at DNA sequence level
can be used as molecular marker for evaluation of phenotypic viability.

• Molecular markers possess unique genetic properties on the basis of

techniques used for detection.

• Two major classes :

hybridization-based markers (RFLP)

PCR-based markers (RAPD)

Restriction Fragment Length Polymorphism (RFLP)

• RFLP, as a molecular marker, is specific to a single clone/restriction enzyme
combination.

• It is difference in homologous DNA sequences that can be detected by the

presence of fragments of different lengths after digestion of the DNA
samples with specific restriction endonucleases

• Most RFLP markers are co-dominant (both alleles in heterozygous sample

will be detected) and highly locus-specific.

• An RFLP probe is a labeled DNA sequence that hybridizes with one or more
fragments of the digested DNA sample after they were separated by gel
electrophoresis, thus revealing a unique blotting pattern characteristic to a
specific genotype at a specific locus.

• Short, single- or low-copy genomic DNA or cDNA clones are typically used
as RFLP probes.

• The RFLP probes are frequently used in genome mapping and in variation
analysis (genotyping, forensics, paternity tests, hereditary disease
diagnostics, etc.).

Developing RFLP probes

 • Total DNA is digested with a methylation-sensitive enzyme thereby
enriching the library for single- or low-copy expressed sequences.

• The digested DNA is size-fractionated on a preparative agarose gel, and

fragments ranging from 500 to 2000 bp are excised, eluted and cloned into
a plasmid vector (for example, pUC18).

• Digests of the plasmids are screened to check for inserts.

• Southern blots of the inserts can be probed with total sheared DNA to
select clones that hybridize to single- and low-copy sequences.

• The probes are screened for RFLPs using genomic DNA of different
genotypes digested with restriction endonucleases.

• Typically, in species with moderate to high polymorphism rates, two to

four restriction endonucleases are used such as EcoRI, EcoRV, and HindIII.

• In species with low polymorphism rates, additional restriction

endonucleases can be tested to increase the chance of finding
polymorphism.

PCR-RFLP

• Isolation of sufficient DNA for RFLP analysis is time consuming and labor
intensive.

• However, PCR can be used to amplify very small amounts of DNA, usually in
2-3 hours, to the levels required for RFLP analysis.

• Therefore, more samples can be analyzed in a shorter time.

• An alternative name for the technique is Cleaved Amplified Polymorphic

Sequence (CAPS) assay.
Applications

• Species Identification

• Genetic variation and population structure study in natural populations

• Comparison between wild and hatchery populations

• Assessment of demographic bottleneck in natural population

• Estimating distances between species and offspring

• Identifying location of QTL's (Quantitative Trait Loci)

• Identification of DNA sequence from useful candidate genes.

Case study: Screening for the sickle-cell gene

• Sickle-cell disease is a genetic disorder in which both genes in the patient

encode the amino acid valine (Val) in the sixth position of the beta chain
(betaS) of the haemoglobin molecule.

• "Normal" beta chains (betaA) have glutamic acid at this position.

• The only difference between the two genes is the substitution of a T for an
A in the middle position of codon 6.
 • When the normal gene (betaA) is digested with the enzyme and the
fragments separated by electrophoresis, the probe binds to
a short fragment (between the red arrows).

• However, the enzyme cannot cut the sickle-cell gene at this site, so the
probe attaches to a much larger fragment (between the blue arrows).

• the pedigree of a family whose only son has sickle-cell disease. Both his
father and mother were heterozygous (semi filled box and circle
respectively) as they had to be to produce an afflicted child (solid box).

• The electrophoresis patterns for each member of the family are placed
directly beneath them. Note that the two homozygous children (1 and 3)
have only a single band, but these are more intense because there is twice
as much DNA in them.

In this example, a change of a single nucleotide produced the RFLP. This is a very
common cause of RFLPs and now such polymorphisms are often referred to
as single nucleotide polymorphisms or SNPs. However, not all RFLPs arise from
SNPs.
How can these tools be used?

• By testing the DNA of prospective parents, their genotype can be

determined and their odds of producing an afflicted child can be
determined. In the case of sickle-cell disease, if both parents are
heterozygous for the genes, there is a 1 in 4 chance that they will produce a
child with the disease.

• Amniocentesis and chorionic villus sampling make it possible to apply the

same techniques to the DNA of a fetus early in pregnancy.
 • The parents can learn whether the unborn child will be free of the disease
or not. They may choose to have an abortion rather than bring an afflicted
child into the world.

Three problems:

• The mutations that cause most human genetic diseases are more varied
than the single mutation associated with sickle-cell disease.

• Over a thousand different mutations in the cystic fibrosis gene can cause
the disease. A probe for one will probably fail to identify a second.

• A mixture of probes, one for each of the more common mutations, can be
used. But there remains the problem of "false negatives": people who are
falsely told they do not carry a mutant gene.

• There are many diseases which result from several mutant genes working
together to produce the disease phenotype.

• There are still genetic diseases for which no gene has yet been discovered.
Until the gene can be located, cloned, and sequenced, no probe can be
made to detect it directly.

• However, it is sometimes possible to find a genetic "marker" that can serve

as a surrogate for the gene itself.

Random Amplified Polymorphic DNA (RAPD)

• It is markers are DNA fragments from PCR amplification of random
segments of genomic DNA with single primer of arbitrary nucleotide
sequence.

How It Works

• Unlike traditional PCR analysis, RAPD does not require any specific
knowledge of the DNA sequence of the target organism: the identical
 primers will or will not amplify a segment of DNA, depending on positions
that are complementary to the primers' sequence.

• For example, no fragment is produced if primers annealed too far apart or

3' ends of the primers are not facing each other.

• Therefore, if a mutation has occurred in the template DNA at the site that
was previously complementary to the primer, a PCR product will not be
produced, resulting in a different pattern of amplified DNA segments on the
gel.

Example

• RAPD is an inexpensive yet powerful typing method for many bacterial

species.

• Silver-stained polyacrylamide gel showing three distinct RAPD profiles

generated by primer OPE15 for Haemophilus ducreyi isolates from
Tanzania, Senegal, Thailand, Europe, and North America.

• Selecting the right sequence for the primer is very important because
different sequences will produce different band patterns and possibly allow
for a more specific recognition of individual strains.

Limitations of RAPD

• Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish
whether a DNA segment is amplified from a locus that is heterozygous (1
copy) or homozygous (2 copies). Co-dominant RAPD markers, observed as
different-sized DNA segments amplified from the same locus, are detected
only rarely.
 • PCR is an enzymatic reaction, therefore the quality and concentration of
template DNA, concentrations of PCR components, and the PCR cycling
conditions may greatly influence the outcome. Thus, the RAPD technique is
notoriously laboratory dependent and needs carefully developed
laboratory protocols to be reproducible.

• Mismatches between the primer and the template may result in the total
absence of PCR product as well as in a merely decreased amount of the
product. Thus, the RAPD results can be difficult to interpret.

Applications

• RAPDs have been used for many purposes, ranging from studies at the
individual level (e.g. genetic identity) to studies involving closely related
species.

• RAPDs have also been applied in gene mapping studies to fill gaps not
covered by other markers.

• DNA finger printing and tagging of markers.

• Variants of the RAPD technique include Arbitrarily Primed Polymerase

Chain Reaction (AP-PCR) which uses longer arbitrary primers than RAPDs,
and DNA Amplification Fingerprinting (DAF) that uses shorter, 5-8 bp
primers to generate a larger number of fragments.

• Multiple Arbitrary Amplicon Profiling (MAAP) is the collective term for

techniques using single arbitrary primers.

Developing Locus-specific, Co-Dominant Markers from RAPDs

• The polymorphic RAPD marker band is isolated from the gel.

• It is amplified in the PCR reaction.

• The PCR product is cloned and sequenced.

 • New longer and specific primers are designed for the DNA sequence, which
is called the Sequenced Characterized Amplified Region Marker (SCAR).

Difference between RAPD and RFLP

Definition:

• If primers with arbitrary sequences are used for amplification, DNA

segments to be amplified wil be selected at random and this provides truly
random sample of DNA markers and so is described Search as random
amplified polymorphic DNA (RAPD).

• RFLP : Restriction Fragment Length Polymorphism.

RAPD - RFLP –
Random Amplified Polymorphic DNA Restriction Fragment Length
Polymorphism

1. Quantity of DNA required for analysis 1. Large quantity of purified DNA

is smal (1050 ng). required i.e. 210 ug
2. Same primers with arbitrary 2. Different species specific probes are
sequence can be used for different required.
species. 3. Comparatively slower processing
3. Fewer steps in procedure therefore it due to more steps involved.
is rapid (Five times quicker than RFLP) 4. Technique comparatively more
4. Technique comparatively less reliable
reliable 5. Can detect allelic variants
5. Cannot detect allelic variants. 6. 13 loci detected
6. 110 loci detected 7. Method involves:
7. Method involves: a) digestion of extracted DNA by
a) extraction of DNA, restriction enzymes,
b) amplification by PCR using b) gel electrophoresis of
random primers, fragments,
c) gel electrophoresis of c) southern blot by specific
amplified DNA and visualisation of probes and detection of specific
markers. sequences
RAPD - RFLP –
Random Amplified Polymorphic DNA Restriction Fragment Length
Polymorphism

REFERENCES

• Brown C. In situ hybridization with riboprobes: an overview for veterinary pathologists. Vet
Pathol. 1998 May;35(3):159-67. PMID: 9598579

• http://www.ncbi.nlm.nih.gov/probe/docs/techrflp

• http://www.ncbi.nlm.nih.gov/probe/docs/techrapd

• http://www.majordifferences.com/2013/02/difference-between-rapd-and-
rflp.html#.VnThEbZ97IV

• Dubey, R.C. 2010. A textbook of biotechnology. S.chand & company LTD : 337-345.