Denaturing High Performance Liquid Chromatography

Principle of DHPLC :

DHPLC ( Denaturing High Performance Liquid Chromatography ) is a chromatographic method allowing detection of substitutions of bases, small délétions or insertions on the level of DNA. Thanks to its raised speed and its resolution, this method is particularly useful for the search for polymorphism S in the DNA.

In practice, the analysis begins with a traditional PCR in order to amplify the studied fragment. If the amplified area has a polymorphism Hémizygote, two types of fragments, corresponding to the wild Allèle and the polymorphic allele, will be present in the product of PCR. This first stage is followed of a stage of denaturation - renaturation in order to create hétéro- and homoduplexes starting from the two populations present in the PCR (Figure I). To seek a polymorphism homozygote, it is necessary to proceed in the same way by mixing as a preliminary a wild population of DNA with a population of polymorphic DNA to obtain hétéroduplexes after the stage of denaturation - renaturation.

Figure I: Creation of the hétéro- and homoduplexes for DHPLC.

The hétéroduplexes are in fact of the double formed bits of DNA of a bit coming from the wild allele and of a bit resulting from the polymorphic allele. The formation of such fragments of DNA then involves the appearance of a “mismatch” or bad pairing at the place where polymorphism is localized.

These “mismatches” present at the level of the hétéroduplexes is at the base of the detection of polymorphisms by the DHPLC. Indeed, the hétéroduplexes, thermically less stable than their homoduplexes corresponding, will be solved thanks to the chromatography when they are subjected to a sufficiently high temperature. The consequence of this instability will be désappariement of the two bits of DNA in the area of polymorphism when the DNA is heated at adequate temperature. This désappariement will consequently involve a reduction in the interaction with the column and thus a time of retention reduced compared to the homoduplexes during chromatographic separation.

To observe this phenomenon of separation, the DHPLC uses a grafted column of a nonporous phase stationary made up of poly (styrene-divinylbenzène) alkylated. This stationary phase is electrically neutral and hydrophobic. The DNA, is negatively in charge to him on the level of its groupings phosphates and cannot thus adsorb itself on the level of the column. In order to return the possible Adsorption, acetate of triéthylammonium (TEAA) is used. The ions ammonium positively charged with these molecules interact with the DNA and the chains alkyl, with the hydrophobic surface of the solid phase.

Thus, when the hétéroduplexes are partially denatured by heating, of the negative charges undergo a partial delocalization and the force of interaction between the DNA of the hétéroduplexes and the column decreases in comparison with the force of interaction of the homoduplexes. The latter will then be élués less quickly by the mobile phase, composed of Acétonitrile, compared to the hétéroduplexes which will then pass the first in front of the Détecteur UV (Ultraviolet) (Figure II).

Figure II: Example of separation of hétéro- and homoduplexes by DHPLC.

See too

HPLC

Random links: