DHPLC is a chromatographic technique for the separation and analysis of DNA fragments with different length and/or base composition. This technique can be applied for mutation detection in DNA fragments of 200–1000 bp in length with a high sensitivity (> 96%) and specificity (> 99%) (3). In addition, the high resolving power of DHPLC allows the distinction of short nucleic acid fragments such as primer extension products for allelic discrimination.
DHPLC is based on a reversed phase system in which the stationary phase is nonpolar and the mobile phase polar (4,5). The hydrophobic stationary phase, DNASep column marketed by the company Transgenomic, is made up of alkylated nonporous poly(styrene-divinylbenzene) particles 2–3 μm in diameter. The polar mobile phase is acetonitrile (CH3-CN). However, DNA molecules are large anions because of the negative charges on the phosphate groups in the phosphate-sugar backbones of the DNA strands.
Organic cations are required to allow interaction between DNA anions and the nonpolar stationary phase. The organic cation carries a positively charged portion to interact with the negative charge of DNA molecules on the one hand, and also a hydrophobic portion to interact with the nonpolar stationary phase on the other hand. The most commonly used organic cation is triethylammonium, (CH3CH2)3N+, in the form of triethylammonium acetate (TEAA). Thus, TEAA is used as an ion pairing reagent. The triethylammonium cations bind to the phosphate groups of DNA molecules and hence effectively coat the DNA molecules with a hydrophobic layer (the triethyl portion).
The number of TEAA molecules coating the DNA molecules is proportional to the length of the DNA molecules and in turn determines the degree of interaction between the DNA molecules and the stationary phase. DNA molecules are eluted from the column in an increasing gradient of acetonitrile, which weakens the interaction between coated DNA molecules and the stationary phase. In other words, coated DNA molecules bind onto the stationary phase and will be released from the stationary phase when acetonitrile in the mobile phase reaches a specific concentration.
Thus, shorter DNA molecules are eluted earlier from the column than and hence separated from longer DNA molecules under the same buffer condition. In summary, the separation of DNA molecules is based on the principle of ion-pair reversed phase liquid chromatography.