Girish Mahajan (Editor)

Denaturing high performance liquid chromatography

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Denaturing High Performance Liquid Chromatography (DHPLC) is a method of chromatography for the detection of base substitutions, small deletions or insertions at the DNA . Thanks to its speed and high resolution, this method is particularly useful for finding polymorphisms in DNA.

In practice, the analysis begins with a PCR standard in order to amplify the fragment studied. If the amplified region exhibits polymorphism hemizygous, two kinds of fragments corresponding to the allele and the wild polymorphic allele will be present in the PCR product. This first step is followed by a step of denaturation - renaturation to create hetero-and homoduplexes from the two populations in the PCR. To find a homozygous polymorphism, proceed in the same way by premixing a DNA wild population to a population of polymorphic DNA to obtain heteroduplexes after the denaturation step - renaturation.

Heteroduplexes are actually double strands of DNA containing a strand from the wild-type allele and a sprig from the polymorphic allele. The formation of such DNA fragments then causes the appearance of a "mismatch" or bad pairing where is located the polymorphism.

These "mismatches" in the heteroduplex present are based on the polymorphism detection by DHPLC. Indeed, heteroduplexes thermally less stable than their corresponding homoduplexes, will be resolved by chromatography when subjected to a sufficiently high temperature. The consequence of this instability will be a mismatch of the two DNA strands in the region of polymorphism when DNA is heated to proper temperature. This mismatch will therefore decrease the interaction with the column and hence a reduced retention time compared to the homoduplexes in chromatographic separation.

To observe the phenomenon of separation, DHPLC using a column of a non-grafted porous stationary phase composed of poly ( styrene - divinylbenzene ) alkyl. The stationary phase is electrically neutral and hydrophobic. The DNA, however, is negatively charged at its phosphate groups and therefore can adsorb by itself at the column. In order to make the adsorption possible, is used of triethylammonium acetate (TEAA). The positively charged ammonium ion of these molecules interact with the DNA and the alkyl chain, with the hydrophobic surface of the solid phase.

So when heteroduplexes are partially denatured by heating, negative charges undergo partial relocation and the interaction force between DNA heteroduplexes and column decreases in comparison to the strength of interaction of homoduplexes. These will then less rapidly eluted by the mobile phase consisted of acetonitrile, heteroduplexes compared to the first pass so that the front detector UV (Ultraviolet).

References

Denaturing high performance liquid chromatography Wikipedia


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