Hello, in this lecture we will be
looking at genetic parentage testing in
animals, specifically the laboratory
procedures for this analysis.
The lecture is part of the Module number
4, Precision Livestock Breeding.
The development of this presentation was
supported by an ERASMUS
+ KA2 grant
within the ISAGREED
project, Innovating the
content and structure of study
programmes in the field of animal
genetics and food resources management
using digitization.
What are the steps of the analysis itself?
First, DNA must be isolated
from the biological material and its
quality verified. Next,
amplify the fragments using
multiplex PCR and separate
the amplicons obtained by
fragmentation analysis. A
special computer program then
automatically compares the results of
the fragmentation analysis
with the calibration curve of the
standard and
determines the alleles of the
individual microsatellites.
After obtaining the genetic profile of
the individual, the parentage
verification is then
performed. Let us now
describe the steps in more
detail.
The first and necessary step in most
molecular genetic methods is DNA
isolation. DNA can be
isolated from almost any
biological material, for example
blood, muscle, buccal swabs,
milk, feathers, hair
or hair bulbs, semen, and so
on. The quality and quantity
of DNA isolated greatly
influences the course of subsequent
reactions. Several
isolation methods exist. The
method is chosen according to the
material from which the DNA is
isolated and the quantity
and quality of the biological
material. At present,
one of the most commonly used methods of
DNA isolation is the spin
column method, which belongs to the
group of methods using solid
particles for DNA isolation.
The advantage of the spin-column method is
primarily speed, simplicity,
high reliability and good
quality and purity of the obtained
DNA. The essence of this
method is the adsorption of DNA onto a
silicate membrane, its washing
and subsequent release into a tube.
The first step is the preparation of the
biological material from which the DNA
will be isolated and its possible
homogenisation. The
images show the most common materials
used for DNA isolation in animals,
for example, blood, tissue, and
hair bulbs, which have the advantage
of easy non-invasive collection
and easy storage and sending of material
from breeders to the laboratory for
analysis.
Lysing buffer and proteinase K are
added to the biological material.
After incubation at an elevated
temperature, a lysate is formed
in which DNA is released from the cell
nucleus and proteins
are degraded.
The resulting lysate is
pipette onto a silicate
matriculate column. During
subsequent centrifugation, the
DNA is captured and bound
to the silicate.
In the next step, the DNA is bound to
the silicate matrix inside the column by
centrifugation. In the next
two steps, the bound DNA is washed
with a wash buffer to remove all
impurities. In the last step,
the purified DNA is released into
the elution buffer solution.
We will use the DNA isolated in this
way for further analysis.
After DNA isolation, it is always
necessary to verify its amount, if the
isolation was successful. The
most common way to verify the amount of
isolated DNA is agarose gel
electrophoresis. In this
case, we will use up a 2%
agarose gel. First,
we weigh 2 grams of agarose.
which is a purified polysaccharide from
seaweed. We boil this
white powder in 100
ml of electrophoretic buffer,
in our case it's TBE buffer,
Tris-borate+EDTA.
This gives us a 2%
concentration of the gel.
Boiling takes place in a microwave
oven until the solution is completely
homogeneous and clear.
Once thoroughly cooked, add the
visualization paint, in this case
GoodView, pour everything
into the prepared tube and
insert the ridges.
Allow the gel to set.
After solidification, the gel
is placed in an electrophoretic bath
filled with electrophoretic buffer,
TBE buffer. The combs are
removed and DNA samples are
applied to the resulting wells.
The DNA must be mixed with a
loading buffer that contains an
indicator dye, usually
bromphenol blue,
and a thickener such as sucrose
or glycerol.
This will allow the DNA to be properly
applied to the bottom of the well.
Each gel must include at least one
column with a size standard.
This is a mixture of DNA of
different and precisely defined
sizes, which is used to
infer or verify the size of the
samples.
After all the DNA samples have been
applied, we connect the
electrophoretic bath to a power
source using electrodes,
set the necessary parameters, in our
case, 130 V
and 30 minutes, and start the
electrophoresis. Once the
separation is complete, we
visualize the result
using UV light. In
the resulting electrophoretogram, we can
see in the first well the
so-called size standard pipetted
for rough quantification of
the results, then in the next
wells, the isolated DNA
of different intensity, different
quantity. After this
electrophoretic verification of the
isolation, the DNA is used
for the next steps of the molecular
genetic analysis.
The last figure of electrophoresis
illustrates the basic steps of
agarose gel electrophoresis.
The next step is a multiplex PCR.
Multiplex PCR is one of many
modifications of the PCR reaction.
The idea is that multiple DNA
fragments are amplified in a single
reaction depending on the number of
primers added to the reaction.
All this takes place in a
thermal cycler. Here
the temperature profile of each
reaction step and the number of
repetitions is set.
The result is a microtube containing
amplicons of different lengths.
The number of amplicons corresponds to
the number of microsatellites
determined in a given individual.
But how to evaluate this reaction? How to
subtract the sizes of the individual PCR
products? Using conventional
agarose gel electrophoresis, the
result is not clearly readable.
Because fluorescently labelled primers
have been added to the reaction,
capillary electrophoresis run in a
genetic analyser can be used to read
the results. Multiplex
PCR is therefore followed by
fragmentation analysis.
The fragmentation analysis itself takes
place in a special genetic analyser.
The analysis is based on capillary
electrophoresis followed by
fluorescence detection of
individual amplified fragments.
The aim is to detect the length of the
DNA fragment, which is defined
by two primers. One
primer is fluorescently labelled,
the other is not. The
advantage of using this method is
that multiple fragments can be analyzed
simultaneously. The
fragments must differ in length or
be fluorescently labelled.
A size standard must be added to each
sample. The operation
of the genetic analyser is similar to
sequencing. And you can see the
demonstration of this instrument in the
Module 1: Sequencing
demonstration.
After the fragmentation analysis is
completed, the results are
evaluated in the special program,
for example GeneMapper.
First, the quality of the raw data needs
to be checked. They look like this.
On the picture, you can see the
resulting electrophoretogram of the set
of 17 microsatellites
in horses. The
individual microsatellites are divided
into four colors in
order to avoid overlapping
alleles and to ensure that the
resulting genotypes are correctly
read and determined. Each
microsatellite consists of one or
two peaks that indicate the
alleles of that microsatellite.
If one allele is present in
the genotype of the selected
microsatellite, the individual
under study is homozygous for that
allele. If the
microsatellite has two alleles
detected, it is heterozygous.
The result of the fragmentation analysis
is therefore the determined
microsatellite set of the
individual. When
verifying parentage in animals,
selected sets of microsatellites of the
parent and offspring are compared.
Based on this, it can then be
determined whether the individual is a
descendant of those parents.
We will illustrate all this with a
concrete example.
On this slide, you can see the resulting
genotypes of the set of 17
microsatellites horses
for the offspring and its possible
parents. The challenge is
to determine if the listed parents
are indeed the parents of the offspring.
If they are, the offspring will have one
allele from the sire and one from the
dam in its microsatellite
genotypes. As we can
see in the offspring, the
genotypes of each locus really
consists of one allele inherited from the
father, marked in blue,
and one allele inherited
from the mother, marked in red.
In this case, genetic testing
confirmed the parentage.
There may be other cases where genetic
analysis rules out one parent
or both parents.
And that's all for this short
presentation explaining genetic
parentage testing in animals. Thank
you for your attention.