Genotyping: Principles and Methods
Genomic selection is based on the estimation of the genetic value of an animal using its SNP marker profile. The method is based on the establishment of a formula for predicting the genetic value from a reference population of several thousand individuals, which are both phenotyped and genotyped. The procedure is based on extracting DNA from blood samples. The entire analysis chain conducted in genetic analysis laboratories for animal species is robotised. The DNA is prepared and then distributed onto a chip on which a scanner enables the genetic markers to be read, using fluorescent reagents.
The Main Stages of Genotyping
Extraction and purification of DNA
DNA can be extracted from any animal tissue. The tissue used most often as a source of DNA is blood, but cartilage, fur, sperm, etc. can also be used. Blood is easy to obtain without an invasive procedure and guarantees a large quantity of DNA compared with other sources. The procedure involves performing a cell lysis initially, and eliminating proteins and other nucleic acids (RNA) by washing reagents. Finally the DNA is concentrated by precipitating it in alcohol. The “jellyfish” is this whitish, translucent, filamentous precipitate made up of long precipitated filaments of DNA.
The analysis methods do not make it possible to appraise all the DNA for a cell. In fact the DNA of a cell contains nearly 3 billion bases in mammals, i.e. a length of about 1.8 m! This is why genotyping seeks to analyse regions or fragments of several hundreds of thousands of bases. So DNA is “fragmented” using restriction enzymes by mechanical fragmentation or irradiation.
Multiplication of DNA
The quantity of DNA can be a limiting factor in the reliability of results. If an interest is taken in a particular fragment of the genome, there are several possible ways of “amplifying” it. The PCR (polymerase chain reaction) method is used. This has three stages:
- The denaturation of two strands of DNA involving separating them into two single strands, often by heating.
- Each strand can be re-paired with its complementary strand or an exogenic complementary strand (depending on the complementarity of the bases). This phenomenon is known as hybridisation.
- Then comes elongation, which corresponds to the synthesis of complementary strands by a polymerase, an enzyme with the role of synthesising strands of nucleotides.
Using this PCR procedure there is a doubling of the number of copies of the amplified fragment with each cycle.
Separation of DNA
The different fragments are separated by electrophoresis, to see them and study them individually.
Several methods are available for revealing DNA fragments
- Staining of the DNA to reveal the fragments after separation
- Prior marking of the DNA fragments by fluorochromes
- Hybridisation of the fragment to be sequenced on oligonucleotides
The DNA Chip (or Microarray), the Ultimate Step
DNA chips are designed using the phenomenon of hybridisation of complementary strands of DNA. Single strand fragments of DNA, for which the sequence is known with the presence of genetic markers, are placed and fixed in an orderly way on glass or silicone slides. The deposit is performed by a robot using micro-drops in “micro-wells”. The fragments thus deposited make up the probe. Depending on the analysis requirements for the chips concerned, hundreds or even thousands or millions of specific fragments may be fixed on the chip medium. The illuminated chip is used most often. It has 54,000 markers. These strands, which were fixed beforehand, are likely to pair by hybridisation with strands contained in the sample of the fragments to be analysed, called the target. The strands of the target are marked according to the analysis that is desired: only the strands carrying the complementary sequence present in the target can hybridise, and the strands that do not contain the sequences sought are removed by washing before the chips are read. The fluorescence of hybridised strands makes it possible to certify the presence of this or that sequence, and consequently this or that allele of the target sample markers.
Reading a Chip with a Fluorescence Scanner
The sample passes through a scanner which then makes fluorescent dots appear on the screen. Each fluorescent dot represents a marker. Each marker reads the form (a or b) of each chromosome of paternal and of maternal origin. As a result the colour shows whether the individual is homozygous (2 identical alleles) or heterozygous (2 different alleles) for this marker.