MOLECULAR

AND
IMMUNOLOGICAL TECHNIQUES
APPLIED IN FISHERIES
I. MOLECULAR TECHNIQUES
A wide range of techniques is now available
for the study of molecular genetics in
fisheries.
Molecular genetic approaches began to be
used in fisheries in the 1950s.
Initial studies were of blood group variants,
primarily in tunas, salmonids and cod.
1. Electrophoresis
Protein or allozyme electrophoresis provides an
indirect assessment of nuclear DNA (nDNA)
variability.
Population structure can be analysed.
The use of allozyme electrophoresis for describing
population structure is probably at its most advanced
stage in the commercial anadromous salmonid fishes.
Hatchery stocks of Atlantic Salmon, have been
reported as having up to 20-30% less heterozygosity
than natural populations .
Direct assessment of DNA variability came with the
isolation of restriction endonucleases.
 These enzymes cut DNA at specific nucleotide
sequences, produce fragments of variable size that can
be separated on electrophoresis gels, and permit the
direct study of DNA sequence variation.
An electric current is applied to the gel, causing one
end of the gel to have a positive charge and the other
to have a negative charge.
All of the restriction fragments begin to move from the
negative end of the gel towards the positive end.
The smaller fragments move faster than the larger
fragments.
After the run DNA fragments have spread out across
the gel, with the smaller ones closer to the positive
end.
 Scientists can identify specific restriction
fragments by their location on the gel.
A complementary sequence of DNA can be used
as a probe to find a restriction fragment on the
gel that has a particular nucleotide sequence.
Most of the initial applications of this technique
was used to examine mitochondrial DNA analysis.
More recently, the development of the
polymerase chain reaction (PCR) enabled minute
quantities of tissue without killing the animal -
For e.g. scales, fins, blood provide sufficient DNA.
2. DNA Fingerprinting
DNA Fingerprinting technique can be used as a powerful
marker system in identification.
Used to verify the identity of cultured cell lines and various
lines of clonal fishes, including those obtained by
gynogenesis and androgenesis.
DNA fingerprints are useful tools in demographic analysis
of fish population.
Their parents can be identified. i.e., identification of
individuals and pedigree.
The fragments detected by DNA fingerprinting can also be
used in gene linkage analysis. If a commercially important
gene tightly linked to a fingerprinting marker, the
transmission of the gene can be determined by inspection
of the marker. This will be of great value in genetic
improvement of fish.
 Fish pathogens can be identified using the DNA
fingerprinting.
Individual specific pattern have been observed in
rainbow trout (O. mykiss), Atlantic salmon, chum
salmon (O. keta), coho salmon (O. kisutch) with
M13 phage or probes.
DNA fingerprinting is useful in annexing paternal
genetic contribution in gynogenetic fish.
Assessment of inbreeding rates,
to study the action of specific genes,
as genetic markers to identify individuals and
family groups and the labelling of broodstocks to
secure ownership property.
3. Dot and slot blotting of DNA
Dot and slot blotting are simple techniques for
immobilizing bulk unfractionated DNA on a nitrocellulose
or nylon membrane.
Hybridization analysis can then be carried out to
determine the relative abundance of target sequences in
the blotted DNA preparations.
Dot and slot blots differ only in the geometry of the blot, a
series of spots giving a hybridization pattern that is
amenable to analysis of by densitometric scanning.
Dot or slot blotting analysis was first developed by Kafatos
et al. (1979).
A large number of samples can be applied at once,
enabling many different DNAs to be sequenced in a single
hybridization experiment.
 The technique has found many application over
the years.
For instance, in genome analysis, information on
the genetic significance of a DNA sequence can
often be obtained by using the sequence as a
hybridization probe to dot blots of DNA prepared
from related species.
The rationale is that most genes have
homologues in related organisms.
For e.g. a coding sequence from the human
genome will probably hybridize to related
sequences in dot blots prepared from DNA of
various mammals.
Gene sequencing
Once an interesting piece of DNA has been
isolated or identified, scientists often need to
determine if the sequence of nucleotides in the
fragment is related to known genes and to
determine what kind of protein it might make.
DNA sequencing is used to detect genetic
mutations linked to diseases such as cystic
fibrosis.
Used to alter the sequence of a gene and study
the function of the resulting protein.
In DNA sequencing, scientists create many
copies of a single-stranded DNA fragment that
will be used to synthesize a new DNA strand.
 An equal number of copies of the fragment are
placed into four different test tubes to act as the
template for the synthesis of a new strand.
The enzyme DNA polymerase and free
nucleotides are added to each test tube.
Each test tube also receives one type of dideoxy
nucleotidea nucleotide that closely resembles
either adenine, guanine, thymine, or cytosine.
These nucleotides can attach to the end of the
new complementary DNA strand, but they
cannot bind to anything else, thus they terminate
the synthesis of the new DNA strand.
 DNA polymerase uses the free nucleotides to
build a complementary DNA strand.
If the original DNA fragment contains guanine,
DNA polymerase delivers a cytosine dideoxy
nucleotide to pair with the guanine base on the
original strand.
The cytosine links with the growing chain of
nucleotides on the complementary DNA strand,
but it is unable to bind with any other nucleotide.
The newly formed DNA fragment terminates with
the cytosine dideoxy nucleotide at the end of the
chain.
 The reactions in each of the four test tubes
produce a series of DNA fragments in which
the new strands terminate at a known base.
Each test tube produces fragments that differ
in length from the other test tubes.
The newly formed fragments are sorted in an
electrophoresis gel that can detect differences
as small as one nucleotide in length.
By analyzing these sorted fragments,
scientists can determine the complementary
base sequence for the original DNA fragment.
Gene chip
The gene chip, also known as a DNA chip or DNA
microarray, is a thumbnail-sized chip of glass or silicon that
carries DNA instead of electronic circuits.
Gene chips can identify the genes that are active within a
cell and help identify mutated genes.
In one application, scientists take a single strand of DNA
that contains a defective gene and use ultraviolet light to
attach the strand onto a glass or silicon chip.
A second DNA strand isolated from a patient is attached to
fluorescent markers and deposited onto the chip.
If the patients DNA strand bonds with the DNA already
bonded to the chip, then the individuals DNA contains the
defective gene.
 When the DNA on the chip pairs with the fluorescent
DNA, it develops a fluorescent glow that can be viewed
with a microscope and interpreted by a computer.
A diagnostic gene chip may soon be manufactured to
hold the DNA sequences of all the known disease-
causing genes, making diagnosis for genetic disorders
fast, reliable, and inexpensive.
Gene chips also distinguish between active DNADNA
that is being transcribed to produce mRNAand
inactive DNA.
Researchers use these chips to learn how the
transcription of a group of genes is affected when cells
are exposed to a drug.
Gene therapy
A recent development in genetic technology known as gene
therapy focuses on curing inherited disorders.
In experiments using gene therapy, researchers have replaced
defective genes with normal alleles, inactivated a mutated gene, or
inserted a normal form of a gene into a chromosome.
The earliest success in human gene therapy involved the treatment
of infants who cannot produce adenosine deaminase (ADA), an
enzyme important to normal function of the immune system.
Scientists have successfully inserted the normal allele for the gene
that codes for the enzyme into cells in ADA-deficient children.
Preliminary evidence indicates that this gene therapy leads to
better immune function in recipients.
Researchers are also exploring gene therapys potential to help
treat people with many other conditions, including certain cancers,
hemophilia, heart disease, and cystic fibrosis.
4. Nucleus transplantation
Studies on nucleus transplantation in fishes were
initiated in the early 1960's in China.
Tong et al. (1963) first demonstrated the
technique, to study the interrelationship
between the cell nucleus and cytoplasm.
The nucleus of a crucian carp egg was removed
with a glass microneedle after removing the egg
capsule with forceps, and put into Holtfreter's
solution in an ice bath.
Then nuclei from the middle or late blastula stage
of the common carp were transplanted into the
enucleated, unfertilized crucian carp eggs.
 In 1973, Tong and Niu, transplanted nuclei
between gold fish (Carassius auratus) and
Rhodeus sinensis for the purpose of studying the
developmental variations between the integrated
nuclei and the pure heterologous nuclei, and the
effects of cytoplasm on the nucleus.
They concluded that character expression (or
genetic expression) was not completely
controlled by the nucleus, or by the cytoplasm.
In fact, it resulted from interactions between
both nucleus and cytoplasm.
 Nuclear-cytoplasmic hybrid fishes have been obtained
from the combination of nucleus and cytoplasm
between two intergeneric species of freshwater teleost
using the technique of electric fusion, i.e. the
combination of the nucleus of carp (Cyprinus carpio
red variety) and the cytoplasm of crucian carp
(Carassius auratus red variety).
Morphological characteristics of those hybrid fish that
have been examined sofar are similar to those of
donor nucleus parental species.
Some of the hybrid fish grow to normal adults. The F3,
F4, F5 descendents have been spread for farm culture.
Protein content of nucleo-cytoplasmic hybrid is 3.78%
higher. Fat content 5.88% lower and the growth rate
15-23 percent faster than those of its parents.
 Chen et al. (1986) transplanted cell nuclei from a grass
carp blastula cell line into unfertilized, enucleated eggs
of crucian carp, thus creating the first " test-tube fish".
They also obtained two fish by transplanting crucian
carp kidney cell nuclei into enucleated crucian carp
eggs, and another three fish by transplanting gold fish
kidney cell nuclei into enucleated crucian carp eggs as
well. These fish grow to reach sexual maturity.
Mao Shujian et al. (1990) transplanted cell nuclei of
the mutant cell line (AHZC- 88), which was resistant to
the grass carp hemorrhagic virus, into unfertilized
grass carp eggs using electric fusion, and raised three
of the fish to the fry stage.
 These examples demonstrate that fish somatic cells
have developmental totipotency.
It shows that many different types of cells have the
ability to develop to the fry stage, and some have
continued to sexual maturity.
Thus, there is a possibility of selecting disease resistant
or cold resistant cell lines for donors and developing a
good strain through nuclear transplantation.
More basic work in the area has been done around
breeding of virus resistant fishes.
Virus-resistant cells have been injected into the eggs
of grass carp, loach and white crucian carp, and a few
eggs have developed to embryos or fry stage
5. Cloning
Genetically identical copies of certain cells and organisms are called
"clones".
Production of clones by two - step gynogenesis: Meiotic and
mitotic gynogenesis.
Through mitotic gynogenesis 100% genetically homozygous clones
are produced in common carp, zebra fish.
Cloning by a combination of andro-and gynogenesis
The production of viable diploid progeny by androgenesis is much
more difficult than through gynogenesis for two reasons.
1. It is difficult to eliminatie female pronucleus and polar bodies
without damaging the cytoplasm.
2. The production of diploid progeny derived from the male
pronucleus is also cumbersome since there is no partner genome
integrating, like the second polar body in gynogenesis.
This is the reason why the first mitotic division has to
be inhibited in androgenesis (endomitosis) for
restoring diploidy. This second step of androgenesis
cytologically corresponds to the whole of mitotic
gynogenesis.
The possible genotypes produced as a result of
successful diploid androgenesis are XX female and
YY super male. In both cases homozygous
individuals are produced, which could be used for
cloning of carp. In the case of common carp female
suppresses the male in growing intensity.
II. IMMUNOLOGICAL TECHNIQUES
The interaction of an antibody with an antigen forms the basis of all
immunochemical techniques.
Antibody binds specifically to an antigen at epitope or antigenic
determinant level and this property has been used for development
of immunodiagnosis.
The antigen binding site of an antibody is formed by the variable
regions of the heavy and light chains. The two variable regions are
closely associated and are bound to each other by noncovalent
interactions.
Binding strength of antibody with the antigen determines the success
of all immunodiagnosis. Binding force between antibody and antigen
are non-covalent depending on hydrogen bonding, electrostatic
force, Van der waals and hydrophobic interactions.
These binding forces depend upon pH, ionic strength and
temperature.
The region of an antigen that interacts an antibody is defined as an
epitope.
The size of an epitope is governed by the size of the combining site.
Solid-Phase Assays
There are three classes of solid phase immunoassays,
1) antibody capture assay,
2) antigen capture assay,
3) two-antibody sandwich assay.
In an antibody capture assay, the antigen is attached to a solid
support, and labelled antibody is allowed to bind.
After washing, the assay is quantitated by measuring the amount of
antibody retained on the solid support.
In an antigen capture assay, the antibody is attached to a solid
support, and labelled antigen is allowed to bind.
The unbound proteins are removed by washing, and the assay is
quantitated by measuring the amount of antigen that is bound.
In a two-antibody sandwich assay, one antibody is bound to first
antibody.
The assay is quantitated by measuring the amount of labelled
second antibody that can bind to the antigen.
The Nature of the Solid Support
The chemical nature of the solid support may be glass, nylon,
sepharose, cellulose, cyanogen bromide, polystyrene or
nitrocellulose paper. However, the most common are
polyvinyl or polystyrene plates and nitrocellulose paper. The
plates are generally coated with a material which encourages
the binding of the antigen or antibody.
 ELISA: Enzyme-linked immunosorbant assay is now
becoming widespread in the medical and veterinary fields
for the rapid diagnosis of many infectious diseases.
The ELISA is a serological technique in which antigen (e.g.
virus) in solution is "trapped" by specific antibodies
immobilized on a suitable solid surface and is detected by
the addition of enzyme-labeled specific antibodies which
cause a colour change in a particular enzyme substance.
Recently the use of the ELISA technique for the detection of
IPNV antigen in infected fish and in cell cultures is reported.
The technique was very rapid (taking under 2 h to
perform), specific and easy to evaluate; it does not require
specialized equipment for routine use.
This technique is applied for the detection of certain fish
rhabdovirus in both cell cultures and in fish.
Targets and Probes
DNA hybridization, whether done on solid phase, in suspension or in situ,
requires two strands of DNA the target and the probe.
The target is the sequence of DNA that is to be detected. The probe DNA
has several essential features.
Firstly, it must be highly complementary to the target strand being sought
for. The more nearly complementary (90-100%) the better the bonding,
and the better pick of the target from the other masses of DNA in the
tissue.
Secondly, there must be some way of detecting the probe once it is
hybridized. Traditionally, this has been radioisotope labeling of probe
with S35, P32, H3 or other radioactive elements.
More recently, a number of other detection systems have been used,
including biotinylated probes.
Commercially, the biotinylated probes seems to be the ones of choice, as
it is straightforward to design to detect these probes.
Thirdly, the probe must be in an appropriate length, long enough to
specifically detect the target, and short enough that the efficiency of
hybridization is high. Probe lengths of 20-100 base pairs are the most
common.
Thank you