denaturing High Performance Liquid Chromatography

(to identify UCH-L1 regions harbouring potential mutations/ polymorphisms) 2.1.4.1 Basic HPLC Theory

High-Performance (or High Pressure) Liquid Chromatography (HPLC) is a form of column chromatography used to separate, identify and quantify. HPLC employs a column that holds chromatographic packing material (the absorbent or stationary phase), a pump that moves the mobile phases (sample being analysed and the solvent that moves it through the column (the most common are methanol and acetonitrile, but the choice is dependent on the nature of the stationary phase and the sample), and a detector that measures the retention times of the molecules.

In HPLC the sample to be analysed (or analyte) is introduced in small volume to the stream of solvent (mobile phase) and is retarded by specific chemical and physical interactions with the stationary phase as it transverses the column length. The nature of the analyte, stationary phase and the mobile phase composition all effect the degree of retardation. One of the fundamental principles of HPLC is the fact that the time it takes for a specific analyte to elute from the mobile phase (retention time) is a unique identifying characteristic of said analyte. The use of pressure in HPLC increases linear velocity through the column, thus giving the component s less time to diffuse, leading to improved resolution in the resulting chromatogram.

Varying the mobile phase composition during the analysis is typically employed and is known as ‘gradient elution’, in which the gradient separates the analyte mixtures as a function of the affinity for the current mobile phase composition relative to the stationary phase (e.g. in a water/ methanol gradient, the more hydrophobic components will elute under relatively high methanol conditions (hydrophobic), whereas the more hydrophilic components will elute relatively low methanol (high water) conditions).

Optimum HPLC methods for a given analyte are those which give the best separation of peaks on the resulting chromatogram.

2.1.4.2 dHPLC Theory

Individuals who are heterozygous in a mutation or polymorphism have a 1:1 ratio of wild-type and mutant DNA. A mixture of hetero- and homoduplexes is formed when the PCR product is hybridised by heating to 95°C and slowly cool ed. After this treatment, a sample will contain a mixture of hetero- and homoduplexes (Figure 15).

Figure 15 – Creation of a mixture of hetero- and homoduplexes through hybridisation (Taylor et al. (1999)).

Mutations are visualised as a characteristic pattern of peaks corresponding to the mixture of heteroduplexes and homoduplexes formed when wild-type and mutant DNA are hybridised.

Figure 16 (refer below) shows the behaviour of four hybridised species in response to a range of temperature regimes used in an experiment. Under the non-denaturing conditions used for separating the DNA fragments (51°C), all f our species have the same retention time. As the temperature increases to 54°C, the heterodupl ex DNA fragments start to denature in the region either side of the mismatched bases and begin to emerge ahead of the still intact homoduplexes. At 56°C the homoduplexes start to den ature, with the A-T homoduplex being marginally more denatured than the C-G homoduplex. Optimum separation is seen to be at 57°C.

Figure 16 – Temperature-dependent resolution of heteroduplexes from homoduplexes. In the absence of the mutation, only one peak, that of the wild-type homoduplex, would be observed (Taylor et al. (1999)).

2.1.4.3 Setting up a dHPLC Method in ‘WavemakerTM’ (Transgenomic, Inc)

- Required DNA sequence was inserted into the DNA sequence panel.

- A helical fraction vs. temperature graph could then be calculated. From this graph, the optimum temperature for each major melting domain (segment of DNA with a difference in helical fraction vs. temperature characteristic) of the DNA sequence was identified – between 75 and 99% helical fraction.

- Method(s) could then be built for each respective melting domain of each DNA sequence to be dHPLC scanned (entire DNA sequences could be realised within 75 and 99% helical fraction at the same temperature).

- The ‘Gradient Template’, which creates the gradient of Buffer A and Buffer B (refer to appendices); critical in the differing elution of heteroduplexes from homoduplexes, and thus their resolution, was then modified in the ‘Gradient Parameters’ panel as follows:-

Drop for Loading – 5%

- Allows non-retained components such as dNTPs, primers and buffers to pass through the separation cartridge when the PCR products are injected, so they do not influence the separation.

Loading Duration – 0.1 mins

Sets the time over which the ‘Drop for Loading’ increases (% Buffer B), to the start of the linear gradient.

Gradient Duration – 4 mins

Length of time over which the % Buffer B increases linearly at a % Buffer B per minute. (Selected by ‘Slope’ option – default of 2% used)

Clean Duration – 0.5 mins

Length of time at which the % Buffer B remains at 100% to clean high molecular weight DNA such as genomic DNA and contaminants from the cartridge.

Equilibration Duration - 20 mins

Length of time that the % Buffer B remains at the initial ‘Drop for Loading’ percentage, to equilibrate for the next injection.

- The rest of the gradient parameters were left at the default values, as they were not deemed important in the dHPLC application used.

- Finally the ‘Acquisition Time’ (time of individual dHPLC scan) was set to 6.8 mins.

- The ‘Wavemaker’ method was then saved and exported for running into ‘HSM’ (main operating software for the dHPLC apparatus – Transgenomic, Inc).

2.1.4.4 dHPLC Protocol

Before the samples could be placed into the ‘auto-sampler’ for the dHPLC mutation scanning run to commence, the following preparatory steps had to be carried out:-

- Buffers A, B and the Wash Solution (Buffer D) were made up fresh every 7 days. Buffer C (75% acetonitrile (Sigma)) was renewed every 3-4 weeks.

- Pumps A, B, C and D were purged for 3-4 minutes to remove any air build up from the system.

- Autosampler line was washed 5 times to remove any air build up.

- Column equilibration took place for 10 minutes at a flow rate of 0.9ml/ minute.

- ‘Rack Parameters’ (dimensions of sample well plate) for 96 well plate containing PCR products were checked in ‘HSM System Manager’ software.

- Sample table was prepared according to PCR products to be scanned and ‘Wavemaker’ methods required. 8µl was entered as amount of sample to be injected, and a 3 minute equilibration time was also entered after each different method to be used in the sample table. - A 75% acetonitrile (Aldrich) wash to clean the separation cartridge, followed by a low flow (0.05ml/ minute – ‘sleep mode’) method were always entered into the sample table to end a dHPLC scanning run.

- In the ‘Autosampler Set Up’ the ‘needle down speed’ was set to ‘Fast’, the ‘injection method’ was set to ‘Cut’, and the ‘lead’ and ‘rear volumes’ were set to 1.0µl.

Once the preparatory steps above had been completed, the samples (in a 96 well plate), which had been heated to 95°C and slowly coole d to allow heteroduplex formation in any samples with a mutation/ polymorphism, were then placed in the autosampler in the pre- specified positions. The dHPLC run was then initialised.