( Most Basic Recommended C...)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 column.

There are columns on the market for performing fragment analysis, they include the DNASep Column, the Helix DVB column and the Zorbax Eclipse dsDNA Analysis Column.

The ion-pairing agent used with either column is triethylammonium acetate (TEAA), which mediates binding of DNA to the stationary phase. Acetonitrile is used as an organic agent to achieve subsequent separation of the DNA from the column.

Typical Method:

Using the system described above, the following reagents and column can be used
• Standards of ds DNA
• Deonised water
• HPLC grade acetonitrile
• Buffer A, comprising of 100 mM TEAA to pH 7.0, 0.1 mM EDTA,
• Buffer B, comprising of 100 mM TEAA to pH 7.0, 0.1mM EDTA and 25 %v/v acetonitrile
• Helix DVB column, 3mm ID x 50 mm length

Set the detection wavelength to 260 nm.

A typical binary gradient cycle is:
A flow rate of 0.45 ml/minute
Percentage of buffer A, when time is 0.00 minute – 55%
Percentage of buffer A, when time is 0.30 minutes – 50%
Percentage of buffer A, when time is 6.00 minutes – 32%
Percentage of buffer A, when time is 7.00 minutes – 32%
Percentage of buffer A, when time is 7.05 minutes – 55%

Samples and standards may need to be diluted with deionised water.

Run the standard to ensure that the required peaks are well resolved. The column may need to be washed, or a wait may be required for the column to equilibrate to the required temperature.

Between changes in temperature, always ensure that the column has had sufficient time to equilibrate to the required temperature.

Optimisation of the denaturing temperature can be performed by trial or error. Here injections of the homozygous sample at temperatures from 52 to 67 °C are made. The retention time of the resulting single peak should increase as the temperature is increased. At the denaturing temperature, the retention time will decrease with the increase in temperature.

Optimisation of the DHPLC temperature may also be determined mathematically, using Stanford University’s Melt Calculator http://insertion.stanford.edu/melt.html.

Check for successful PCR amplification by running the homozygous sample under non denaturing conditions , ie less than 50 C and then at the optimal DHPLC temperature. If the PCR conditions have been optimised then a single peak will be seen.

Run the heterozygous sample at the optimum DHPLC temperature. More than one peak should be seen.

If the heterozygous sample does not give a significantly different peak pattern than that seen for the homozygous sample, increase or decrease the column temperature until a different pattern is seen.

Typical Chromatograms:

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