Hello, in this presentation, we will be
looking at genomics in livestock and in
particular sequencing. We will use the
example of the laboratory process of
Sanger sequencing on an automatic
sequencer. This presentation is part
of module 1, Animal Genetics. The
creation 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.
The actual laboratory procedure of Sanger
sequencing involves several steps.
The first is the preparation of a suitable
sample, i.e. DNA. The
sample can be DNA cloned in a vector
and obtained by isolation from bacterial
cells. Universal primers are
used for sequencing and therefore, the
result is usually of high quality.
However, cloned DNA is not
always available. More often, we use
the PCR product as template.
Here, the purity of the PCR product is
critical (the PCR product must be
purified), as well as
its quantity. If we have a good quality
sample, we can proceed to the second
step, the actual sequencing reaction.
This is an enzymatic reaction similar to
PCR, but only one primer
and commercially available reaction
mixture containing specific fluorescently
labeled ddNTPs (each
base a different color), colled
terminators, must be used.
The result is a mixture of fragments,
each terminated by a terminator, which
indicated by its collour the base at
the corresponding site in the sequence.
For a detailed description of the method,
see the lecture on this topic.
This is followed by removal of the
free nucleotides
and precise capillary electrophoresis.
The final step is the software evaluation
of the data and assembly of the
final sequence.
One option for removing free
nucleotides, especially free clolor-labeled
terminators, is to use an
emulsion of a porous reagent that
absorbs these nucleotides and thus
purifies the solution. The reagent
must be thoroughly remixed by vortexin
before adding it to the sequencing
mixture and than removed with a sheared
tip.
To do this, the sequencing mixture and
emulsion should be allowed to shake for
30 minutes.
After centrifugation, the
supernatant is removed into a sequencing
plate and is ready for analysis in
the sequencer.
The basis of the automatic sequencer is
a fluorescence capillary ectophoresis.
This picture shows the basic components
of the instrument: the anode section
with the polymer pump on the left, the
detection chamber in the middle, the
capillary array on the top right, and
the sample plate and the cathode section on
the bottom. Prior to each
separation, the pump pushes fresh
polymer into the capillaries, then the
sample is electro-injected into the
capillary, and separation proceeds
from the cathode to the anode.
The fluorescent signal is picked up in
the detection section, with shorter DNA
fragments arriving before longer
ones.
This picture shows a close-up of the
pump, polymer bag, anode
and anode buffer container.
Glass capillaries filled with polymer are
the basis of precise DNA
separation. Polymer is a
special gel with separation
ability. The DNA travels through the
capillary and separates according to
size. Smaller fragments reach
the window earlier than longer ones
and the instrument reads the
corresponding signal (fluorescent
color).
Insertion of fresh anode
electrophoresis buffer must be done
very carefully. You can see the
block with the pump, which always fills
the capillary with fresh polymer before the
run. The bag on the right contains
a separation polymer.
Auto sampler extends and is ready to
load the sample plate.
Snap the plate retainer (cover)
onto the plate, septa, and plate
base.
First, we insert a cartridge with cathode
buffer (left side)
and washing solution (right)
and subsequently plate assembly.
After closing, the sequencer will
automatically move to the correct
position.
We can insert a maximum of two
plates with samples, i.e. a total
of 192
samples.
After the wasching step,
the electrodes are immersed in the
samples and electro-injection into the
capillaries take place.
Then the electrodes are placed in the
cathode buffer and electrophoresis
begins.
The reading of the fluorescent signal
takes place gradually as the relevant
fragments turn to the sensor.
After the electrophoresis is finished,
we will check the raw data.
The evaluation is carried out using
sequencing analysis software. The
color of the peak corresponds to the
corresponding base in the sequence shown
above. Here we also see
an example of a heterozygote.
It can be recognized by the fact that
there are peaks of two different colors
at the same position.
Specific software can be used to compare
the sequences of a large number of
samples and thereby find
differences: polymorphism.
In the mentioned bee samples,
there are two types of polymorphism:
division on the left and single
nucleotide on the right.
Evaluation of this result allowed us to
distinguish different mitochondrial
DNA haplotypes in our bee
population that are related to their
origin.
We can export a finished sequence
in a suitable format for further use.
For comparing finished sequences,
e.g., the Clustal program is suitable.
We can thus find disease-causing
mutations, alleles influencing
performance traits in farm animals,
determine genetic diversity in
animal populations, kinkship, and
many other applications.
And that's all for this short
presentation explaining the basics of
DNA sequence determination. I
believe that the illustrative examples of
this video will help you understand one
of the basic laboratory methods
without which modern genetics and
genomics cannot be. Thank you for
your attention.