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Denaturing HPLC (DHPLC)


Most Basic Recommended Cecil Instrument:
Cecil Biocompatible HPLC System 4, plus column oven, plus an optional biocompatible autosampler which will accommodate 96 well plates. A biocompatible installation kit is required. A fraction collector may be added.


Introduction:
DHPLC is an ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) technique.

It identifies mutations and polymorphisms based on detection of heteroduplex formation between mismatched nucleotides in double-stranded PCR amplified DNA. Sequence variation creates a mixed population of heteroduplexes and homoduplexes during reannealing of wild-type and mutant DNA. When this mixed population is analysed by HPLC under partially denaturing temperatures, the heteroduplexes elute from the column earlier than the homoduplexes because of their reduced melting temperature.

Optimisation of DHPLC, requires the generation of a melting profile for each PCR fragment to determine its optimal temperature for analysis.

Usually, the lowest temperature that shows a change in retention time of 1 minute is optimal for identification of sequence variation.


Minimum Analytical Instrument Requirement:

DHPLC requires
• A biocompatible HPLC gradient pumping system with a flow path composed of PEEK tubing fittings, and filters
• An ultraviolet (UV) detector capable of 254 or 260 nm
• A column oven capable of maintaining temperatures of 50°C to 80°C.
• An autosampler configured to handle 2 x 96 0.2-mL microtubes is optimal for automated processing of samples.


An optional fraction collector can be added to the system to automate the isolation and purification of fragments as they elute from the col
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IC50: 3 ng/ml · Limit of detection: 0.9 ng/ml...
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