Denaturing DHPLC

DHPLC (C2, L3, U1) uses a proprietary matched ion polynucleotide chromatography column devised by Transgenomics, Inc., in combination with an organic solvent and thermal denaturation (rather than an electrophoretic gel) to resolve heteroduplexes containing a suspected polymorphism from a polymorphism-free homoduplex. Homo- and heteroduplexes are visualized through UV absorbance readings of the eluent as it leaves the column. 

Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN, James CD (1998) Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res 26 1396-1400... 

DHPLC denaturing high-pressure liquid chromatography... 

Interestingly the temperature was a crucial parameter to achieve DNA [32-35] and RNA denaturation [36] (see DHPLC in Chapter 16 for further details). 

FIGURE 16.1 Steps of denaturing high performance liquid chromatography (DHPLC). 

SSCP, single-strand conformation polymorphism PNA-LNA, peptide nucleic acid-locked nucleic acid MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ARMS, amplified refractory mutation system dHPLC, denaturing high performance liquid chromatography. 

One specialty in dehybridization detection is that of dHPLC (denaturing high-performance liquid chromatography). Hybridization products differ in melting temperature and mobility on a specific support if a mismatch is introduced by a SNP. In the case of a SNP, a heteroduplex is formed in the analyzed sequence, whereas in the case of identical sequences (no SNP present), a perfect homoduplex is formed. These events can be discriminated by dHPLC at a low to medium throughput for SNP discovery or SNP detection. 

Denature samples at 96°C for 4 min followed by 4°C hold before running the samples on DHPLC instrument. 

In instances in which SBE reactions are pooled prior to running on the DHPLC, a minimum of 8 pL of each individual reaction are combined prior to denaturation (see Note 23). 

PCRs can be performed and stored at 4°C in advance however, it is essential to denature and reanneal the samples just prior to loading samples on the DHPLC instrument. 

Frueh, F. W. and Noyer-Weidner, M. (2003) The use of denaturing high performance liquid chromatography (DHPLC) for the analysis of genetic variations impact for diagnostics and pharmacogenetics. Clin. Chem. Lab. Med. 41,452-461. 

The usefulness of LIE in DNA analysis has been demonstrated using fluorescent dyes as labels in CE and denaturing LC (DHPLC) techniques. It has been suggested that CE is preferable for DNA sizing and sequencing analyses and DHPLC is effective for screening of genetic mutations and polymorphisms. 

Hutzler, M., Geiger, E., Jacob, F. (2010). Use of PCR-DHPLC (Polymerase chain reaction-denaturing high performance liquid chromatography) for the rapid differentiation of industrial Saccharomyces pastorianus and Saccharomyces cerevisiae strains. Journal of the Institute of Brewing, 116, 464-474. 

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