Denaturing High Performance Liquid Chromatography (DHPLC) is a method of

chromatography In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system (a ...

for the detection of base substitutions, small deletions or insertions in the DNA.{{Cite journal , last=Xiao , first=W. , last2=Oefner , first2=P. J. , date=June 2001 , title=Denaturing high-performance liquid chromatography: A review , url=https://pubmed.ncbi.nlm.nih.gov/11385705/ , journal=Human Mutation , volume=17 , issue=6 , pages=439–474 , doi=10.1002/humu.1130 , issn=1098-1004 , pmid=11385705, doi-access=free Due to its speed and high resolution, this method is particularly useful for finding polymorphisms in DNA. In practice, the analysis begins with a standard

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...

(PCR) in order to amplify the fragment of interest. If the amplified region that exhibits the polymorphism(s) is

heterozygous Zygosity (the noun, zygote, is from the Greek "yoked," from "yoke") () is the degree to which both copies of a chromosome or gene have the same genetic sequence. In other words, it is the degree of similarity of the alleles in an organism. Mo ...

, 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 allele 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–renaturation step. 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 the polymorphism is located. These "mismatches" in the heteroduplex are the basis for the polymorphism detection by DHPLC. Heteroduplexes are thermally less stable than their corresponding homoduplexes, and the single DNA strands will therefore be disconnected by chromatography when subjected to a sufficiently high temperature. The consequence of this double strand instability will be a mismatch of the two DNA strands in the region of polymorphism when DNA is heated to the DNA

melting Melting, or fusion, is a physical process that results in the phase transition of a substance from a solid to a liquid. This occurs when the internal energy of the solid increases, typically by the application of heat or pressure, which incre ...

temperature. This mismatch will therefore decrease the interaction with the column and will result in a reduced retention time compared to the homoduplexes in the chromatographic separation process. To observe the phenomenon of separation, the DHPLC method uses a

column A column or pillar in architecture and structural engineering is a structural element that transmits, through compression, the weight of the structure above to other structural elements below. In other words, a column is a compression member. ...

of a non-grafted porous stationary phase composed of

polystyrene Polystyrene (PS) is a synthetic polymer made from monomers of the aromatic hydrocarbon styrene. Polystyrene can be solid or foamed. General-purpose polystyrene is clear, hard, and brittle. It is an inexpensive resin per unit weight. It is a ...

divinylbenzene Divinylbenzene (DVB) consists of a benzene ring bonded to two vinyl groups. It is related to styrene (vinylbenzene) by the addition of a second vinyl group.CRC Handbook of Chemistry and Physics 65Th Ed. It is a colorless liquid manufactured by ...

alkyl. The stationary phase is electrically neutral and

hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, th ...

. The DNA, however, is negatively charged at its phosphate groups and therefore can adsorb itself on the column. In order to make the

adsorption Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface. This process creates a film of the ''adsorbate'' on the surface of the ''adsorbent''. This process differs from absorption, in which a f ...

possible, triethylammonium acetate (TEAA) is used. The positively charged ammonium ion of these molecules interacts with the DNA, and the alkyl chain with the hydrophobic surface of the solid phase. Therefore, when heteroduplexes are partially denaturated by heating, the negative charges undergo partial relocation and the interaction force between DNA heteroduplexes and column decreases in comparison to the strength of interaction of the homoduplexes. These will therefore be eluted less rapidly by the mobile phase (consisting of

acetonitrile Acetonitrile, often abbreviated MeCN (methyl cyanide), is the chemical compound with the formula and structure . This colourless liquid is the simplest organic nitrile (hydrogen cyanide is a simpler nitrile, but the cyanide anion is not clas ...

References

Chromatography