Direct amplification :
It is possible to amplify a particular DNA fragment. The specificity is brought by two small DNA pieces (names primers), known to be part of the fragment to be amplified.
These primers associate against the DNA strand at the complementary place of the genetic code. An enzyme, named DNA polymerase, reconstitute the DNA between the two primers.
This generates a copy of the original fragment. This operation is repeated many times starting with the fragment and the copy and allows to obtain many copies.
This is the technique named polymerase chain reaction (PCR). It is then easier to identify the fragments which size may be measured by electrophoresis.
Indeed, the fragments of known size are obtained only if the primers have been associated and the primers have been associated only if the corresponding genetic code was on the original fragment.
So, the specific primers of a species to be searched will amplify a fragment of known size only the species DNA is present in the sample. |
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Sequencing :
When two genetic entities to be identified have very close DNA, it is impossible to find specific primers to everyone.
Common primers to both entities are then used to generate a DNA fragment copy but the identification is done by the reading of small differences in the genetic code.
This may be done using the sequencing that allows to obtain the genetic code written on the fragment to be compared to a known referenced sequence.
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Real-time fluorimetry and quantification :
When DNA is amplified, it is possible to use two more primers that will generate a fluorescent signal, either when they associate to the strand or after association, when the polymerase works. The more on going DNA copies are present, the more fluorescent primers are associated, the more fluorescence will be emitted. Furthermore, the more DNA is present at the beginning, the more fluorescence is available and measurable.
This principle allows the DNA quantification in a sample for example, it is used for the quantification of the DNA GMO percentage.
The quantification is done on a real time fluorescent thermocycler.
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