WHAT IS DNA                                                                                                             Figure 1

DNA ( which is short for deoxyribonucleic acid ) is the genetic material found              

in the cells of most living organisms, including human beings. It forms the blueprint

used to build that organism and controls its distinguishing characteristics and features.

As it is the DNA within each individual that mostly determines their appearance, 

and DNA is passed from parent to offspring, offspring tend to look like their parents.

 

A good way to think about the structure of  DNA is to imagine a spiral staircase

(see Figure 1). This staircase is very, very long, indeed if the distance between each

step was 12 inches the human DNA would reach the moon and back.

                                                                                                                                        Chromosome                   DNA spiral

                                                                                                                                                                                 'staircase'

HOW IS DNA ORGANISED ?

The DNA staircase of an organism is divided into a number of separate segments known as chromosomes. All animals have a specific number of chromosomes. For example, dogs have 78, cows have 60 and humans have 46. The chromosomes are always arranged in matched pairs. For example, the 46 chromosomes in human cells are arranged in 23 matched pairs- one pair of sex chromosomes (x and y) and 22 other pairs known as autosomes (see Figure 2). One  chromosome from each of these matched pairs is inherited from the mother and the other from the father.

                                                Figure 2

Diagram of the complete chromosome complement for a human male. All this DNA is packed into a space smaller than the point of a needle.

SO WHAT IS GENOTYPING ?                                                                                                                             

If we think of the chromosome as a long DNA spiral staircase that connects all the floors of a very tall building, then genotyping can be thought of as the measurement of the distance any two of these floors.

The space between each floor is called a  locus  (Latin for location). To make it clear which part of the staircase we are measuring, the locus is given a specific code, eg, ABC123 (see Figure 3). The height height of the staircase can then be measured at this particular location.

Now, because the chromosomes are in pairs (see Figure 2), there are actually two staircases to measure at any one locus. But, and this is the interesting property of DNA, the length of the staircase between any two floors is not necessarily the same for each one of the chromosomes in the pair. So, while the staircase at one locus for one chromosome could be measured at 20 steps, the same locus for the other chromosome could be measured as 30 steps. The combination of these two lengths is what is what is termed the genotype for that locus, which in this case would be 20/30.

However to make the genotype easier to understand, we use letters rather than numbers, with each letter representing a specific length of staircase.

For example, in Figure 4, we can see that Dog 1 has two staircases of the same length-20 steps. If we use 'B' to represent 20 steps, then the genotype at locus ABC123would be B/B On the other hand, Dog 2 has one staircase that is 20 steps and the other is 30 steps, so if we use 'C' to represent 30 steps this genotype would be reported as B/C.

Figure 4

Figure 3

HOW DO YOU IDENTIFY AN ANIMAL USING A GENOTYPE ?

The genotype of an animal is fixed at the moment of conception and remains constant throughout their life. This means that if an animal is genotyped at any point in its life, or indeed, after death, the genotype obtained will be the same.

In addition, regardless of which kind of sample is used for the analysis-blood, hair, cheek cells, milk or semen-the genotype will be identical.

As explained, the length of the staircase is not necessarily the same for each one of chromosomes in a pair.  However, there are actually only a limited number of length variants that can be detected in any group of individuals. For example, locus ABC123 may have only 10 different lengths of staircase available. This means that only the first 10 letters of the alphabet are necessary to represent all the possible genotypes at this locus. As there are only 10 variants, or alleles to give them their scientific name, at this locus it is probable that two separate animals will have the same genotype.

So, to increase the discrimination power of genotyping, more loci (the plural of locus) are measured, as although the genotype at a single locus is not unique to an individual, the combination of genotypes at a range of loci can be. As each of these loci has a number of alleles (more than 10 in most cases) the greater the number of separate loci examined the smaller the chance that two individuals will have the same overall genotype. For example, assume that the probability of any one particular genotype (that is, combination of letters) occurring at any specific locus is 1 in 10. Then, if two loci were examined, the overall probability of finding two individuals with identical genotypes would be 1 in 100 (1/10 x 1/10). If three loci were examined the probability would become 1 in 1000 (1/10 x 1/10 x 1/10) and four loci would become 1 in 10,000 (1/10 x 1/10 x 1/10 x 1/10), and so on.

In most genotyping work at least 10 different loci are routinely examined and measured. the result of the analysis is recorded in the form of a certificate of identification as shown in Figure 5.

 

 Figure 5

In Table 1 below, for example, you can see the genotype of three different dogs across three loci called ABC123, DEF345 and GHI777.From this we can tell that Prince, Rover and Frisky all have the same genotype at locus ABC123, so they cannot be individually identified by this one locus. However, when the results of locus DEF345 are examined, it can be seen that Frisky has a different genotype to the other two so he can now be genetically identified.

This still leaves Prince and Rover who require further analysis in order to distinguish them. The examination of a third  locus, at GHI777, provides the answer we need. Both these dogs have different genotypes at this locus so all thee dogs can now be individually identified using their genotype.
Table 1

HOW DO YOU USE GENOTYPING FOR PEDIGREE IDENTIFICATION?

DNA is divided into segments called chromosomes that exist as matched pairs, as shown in Figure 2. One of each pair is from the mother and the other from the father,  which is why there are always two alleles to measure at each specific locus.

When we want to verify the parentage of an animal the genotype of the offspring is compared to those of the parents. As it is the biological parents who contribute all the genetic material found in an offspring, the specific letters in the offspring's genotype must also be present in the parents genotypes for the parentage to be possible. This situation is termed an inclusion. If, however, some or all of the letters in the offspring's genotype are not present in at least one of the parental genotypes, then the result is termed an exclusion and the parentage is not possible.

Figure 6 illustrates an inclusion. Here we are looking at canine parentage verification using just one locus for simplicity. We normally compare the mother with the offspring first as maternity is more likely to be correct than paternity. First we look at the pup's genotype then we look at the mother's. The common letter is "M", so we can say that the pup has inherited the allele "M" from mother. The remaining letter in the pup's genotype, "A", must, therefore be inherited from the true biological father so we look at the presented father's genotype to see if the letter is there. In this case it is, so we can confirm that the presented mother and father are the true biological parents of the puppy.

Figure 6
Figure 7

In Figure 7 we use cattle to show an exclusion, again using just one locus for simplicity.

As with the inclusion, we compare the mother with the offspring first. In this case the "M" is the maternally - contributed allele. The remaining letter in the calf, "A", must be paternally - contributed. However, this time the presented father has a genotype of "C/D", so he cannot be the true biological father of the calf.

WHY SHOULD WE  DNA PROFILE OUR DOGS ?

   You can be protected - both as a breeder and a buyer - and add value to your dog.

   You can find out the true sire of any individual dog.

   If your dog is lost or stolen you will be able to re-identify it - without any doubt

   For the first time you can now confirm the pedigree of your dog.

INFORMATION COURTESY OF ROSGEN LTD

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