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Hello, in this lecture we will be

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looking at genetic parentage testing in

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animals, specifically the laboratory

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procedures for this analysis.

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The lecture is part of the Module number

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4, Precision Livestock Breeding.

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The development of this presentation was

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supported by an ERASMUS

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+ KA2 grant

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within the ISAGREED

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project, Innovating the

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content and structure of study

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programmes in the field of animal

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genetics and food resources management

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using digitization.

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What are the steps of the analysis itself?

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First, DNA must be isolated

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from the biological material and its

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quality verified. Next,

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amplify the fragments using

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multiplex PCR and separate

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the amplicons obtained by

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fragmentation analysis. A

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special computer program then

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automatically compares the results of

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the fragmentation analysis

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with the calibration curve of the

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standard and

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determines the alleles of the

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individual microsatellites.

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After obtaining the genetic profile of

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the individual, the parentage

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verification is then

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performed. Let us now

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describe the steps in more

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detail.

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The first and necessary step in most

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molecular genetic methods is DNA

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isolation. DNA can be

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isolated from almost any

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biological material, for example

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blood, muscle, buccal swabs,

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milk, feathers, hair

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or hair bulbs, semen, and so

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on. The quality and quantity

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of DNA isolated greatly

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influences the course of subsequent

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reactions. Several

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isolation methods exist. The

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method is chosen according to the

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material from which the DNA is

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isolated and the quantity

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and quality of the biological

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material. At present,

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one of the most commonly used methods of

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DNA isolation is the spin

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column method, which belongs to the

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group of methods using solid

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particles for DNA isolation.

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The advantage of the spin-column method is

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primarily speed, simplicity,

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high reliability and good

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quality and purity of the obtained

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DNA. The essence of this

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method is the adsorption of DNA onto a

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silicate membrane, its washing

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and subsequent release into a tube.

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The first step is the preparation of the

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biological material from which the DNA

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will be isolated and its possible

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homogenisation. The

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images show the most common materials

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used for DNA isolation in animals,

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for example, blood, tissue, and

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hair bulbs, which have the advantage

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of easy non-invasive collection

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and easy storage and sending of material

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from breeders to the laboratory for

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analysis.

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Lysing buffer and proteinase K are

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added to the biological material.

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After incubation at an elevated

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temperature, a lysate is formed

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in which DNA is released from the cell

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nucleus and proteins

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are degraded.

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The resulting lysate is

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pipette onto a silicate

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matriculate column. During

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subsequent centrifugation, the

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DNA is captured and bound

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to the silicate.

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In the next step, the DNA is bound to

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the silicate matrix inside the column by

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centrifugation. In the next

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two steps, the bound DNA is washed

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with a wash buffer to remove all

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impurities. In the last step,

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the purified DNA is released into

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the elution buffer solution.

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We will use the DNA isolated in this

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way for further analysis.

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After DNA isolation, it is always

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necessary to verify its amount, if the

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isolation was successful. The

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most common way to verify the amount of

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isolated DNA is agarose gel

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electrophoresis. In this

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case, we will use up a 2%

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agarose gel. First,

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we weigh 2 grams of agarose.

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which is a purified polysaccharide from

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seaweed. We boil this

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white powder in 100

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ml of electrophoretic buffer,

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in our case it's TBE buffer,

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Tris-borate+EDTA.

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This gives us a 2%

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concentration of the gel.

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Boiling takes place in a microwave

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oven until the solution is completely

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homogeneous and clear.

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Once thoroughly cooked, add the

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visualization paint, in this case

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GoodView, pour everything

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into the prepared tube and

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insert the ridges.

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Allow the gel to set.

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After solidification, the gel

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is placed in an electrophoretic bath

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filled with electrophoretic buffer,

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TBE buffer. The combs are

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removed and DNA samples are

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applied to the resulting wells.

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The DNA must be mixed with a

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loading buffer that contains an

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indicator dye, usually

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bromphenol blue,

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and a thickener such as sucrose

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or glycerol.

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This will allow the DNA to be properly

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applied to the bottom of the well.

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Each gel must include at least one

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column with a size standard.

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This is a mixture of DNA of

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different and precisely defined

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sizes, which is used to

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infer or verify the size of the

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samples.

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After all the DNA samples have been

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applied, we connect the

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electrophoretic bath to a power

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source using electrodes,

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set the necessary parameters, in our

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case, 130 V

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and 30 minutes, and start the

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electrophoresis. Once the

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separation is complete, we

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visualize the result

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using UV light. In

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the resulting electrophoretogram, we can

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see in the first well the

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so-called size standard pipetted

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for rough quantification of

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the results, then in the next

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wells, the isolated DNA

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of different intensity, different

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quantity. After this

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electrophoretic verification of the

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isolation, the DNA is used

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for the next steps of the molecular

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genetic analysis.

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The last figure of electrophoresis

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illustrates the basic steps of

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agarose gel electrophoresis.

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The next step is a multiplex PCR.

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Multiplex PCR is one of many

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modifications of the PCR reaction.

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The idea is that multiple DNA

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fragments are amplified in a single

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reaction depending on the number of

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primers added to the reaction.

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All this takes place in a

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thermal cycler. Here

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the temperature profile of each

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reaction step and the number of

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repetitions is set.

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The result is a microtube containing

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amplicons of different lengths.

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The number of amplicons corresponds to

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the number of microsatellites

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determined in a given individual.

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But how to evaluate this reaction? How to

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subtract the sizes of the individual PCR

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products? Using conventional

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agarose gel electrophoresis, the

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result is not clearly readable.

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Because fluorescently labelled primers

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have been added to the reaction,

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capillary electrophoresis run in a

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genetic analyser can be used to read

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the results. Multiplex

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PCR is therefore followed by

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fragmentation analysis.

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The fragmentation analysis itself takes

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place in a special genetic analyser.

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The analysis is based on capillary

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electrophoresis followed by

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fluorescence detection of

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individual amplified fragments.

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The aim is to detect the length of the

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DNA fragment, which is defined

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by two primers. One

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primer is fluorescently labelled,

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the other is not. The

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advantage of using this method is

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that multiple fragments can be analyzed

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simultaneously. The

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fragments must differ in length or

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be fluorescently labelled.

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A size standard must be added to each

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sample. The operation

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of the genetic analyser is similar to

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sequencing. And you can see the

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demonstration of this instrument in the

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Module 1: Sequencing

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demonstration.

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After the fragmentation analysis is

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completed, the results are

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evaluated in the special program,

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for example GeneMapper.

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First, the quality of the raw data needs

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to be checked. They look like this.

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On the picture, you can see the

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resulting electrophoretogram of the set

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of 17 microsatellites

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in horses. The

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individual microsatellites are divided

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into four colors in

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order to avoid overlapping

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alleles and to ensure that the

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resulting genotypes are correctly

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read and determined. Each

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microsatellite consists of one or

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two peaks that indicate the

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alleles of that microsatellite.

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If one allele is present in

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the genotype of the selected

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microsatellite, the individual

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under study is homozygous for that

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allele. If the

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microsatellite has two alleles

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detected, it is heterozygous.

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The result of the fragmentation analysis

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is therefore the determined

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microsatellite set of the

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individual. When

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verifying parentage in animals,

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selected sets of microsatellites of the

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parent and offspring are compared.

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Based on this, it can then be

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determined whether the individual is a

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descendant of those parents.

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We will illustrate all this with a

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concrete example.

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On this slide, you can see the resulting

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genotypes of the set of 17

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microsatellites horses

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for the offspring and its possible

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parents. The challenge is

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to determine if the listed parents

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are indeed the parents of the offspring.

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If they are, the offspring will have one

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allele from the sire and one from the

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dam in its microsatellite

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genotypes. As we can

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see in the offspring, the

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genotypes of each locus really

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consists of one allele inherited from the

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father, marked in blue,

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and one allele inherited

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from the mother, marked in red.

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In this case, genetic testing

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confirmed the parentage.

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There may be other cases where genetic

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analysis rules out one parent

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or both parents.

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And that's all for this short

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presentation explaining genetic

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parentage testing in animals. Thank

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you for your attention.