Development o f PMD

4.6.1. Temperature control

In all runs to be described, the temperature was measured by means of a thermocouple in the tank reporting back temperature information to the electrophoresis control software. This temperature was presented in two forms on the computer control screen: (1) a desired bath temperature, (2) an actual bath temperature. The desired bath temperature was an indication of the stage reached in the programmed melting gradient and the actual bath temperature was a reading of the buffer and gel temperature at that particular time; this value was in constant fluctuation and reflected the fact that frequent temperature measurements were being taken. This fluctuation was averaged out, and the instrument response was dictated by this average. Equipment performance was also examined using a high accuracy, high precision platinum resistance wire thermometer (M^ Instrumentation, Holbury Southampton). This thermometer had been calibrated between 40°C and 80°C in increments of 10°C. Calibration had been achieved by immersing the probe in closely controlled temperature reference environments together with two reference standard instruments. Accuracy of this thermometer was to within +/- 0.05°C. Any deviation in temperature readings between the platinum resistance thermometer and the thermocouple was adjusted and corrected for using a calibration factor.

4.6.2. Establishing the conditions o f melting fo r exon 3 o f the LDLR gene

Migration velocity of normal and mutant PCR product was determined by a series of different constant temperature runs. The exon 3 mutations used in the development of PMD are shown in Figure 4.2. PCR amplified mutants for exon 3 of the LDLR gene and normal controls were set up as described in section 2.2.3. Gels for PMD analysis were cast and samples loaded in the manner described in sections 2.5.3 and 2.5.4. Several thirty minute constant temperature runs were set up to determine the point at which a transition occurred from native and wound to denatured and unwound or melted. The observed velocity of the DNA was taken as the distance migrated from each well to its position in the gel. Bromophenol blue and xylene cyanol dye migration was also noted for each set of conditions. Thus, a series of 30 min constant temperature experiments were performed where temperatures of 55°C, 60°C, 65°C, 70°C and 75°C were used. Depending on the migrational distance (i.e. the melting) of the DNA, further experimental runs at the intermediate temperatures of 66°C, 67°C, 68°C and 69°C were then carried out to determine the precise temperature that produced the maximal

retardation in velocity. This melt profile was compared with that produced from the program melt87 (Lerman and Silverstein, 1987) and MS melt (Spanakis et a l In Preparation). These programs are based on theoretical models of empirical melting data. On the basis of the observed velocity profile, a real-time-variable-temperature gradient was selected for trial. An increasing temperature gradient was chosen over a range of 8°C, starting several degrees below the stage at which the DNA is in its native unwound form, and finishing 1-2°C above the completely melted form.

4.6.3. P roof o f principle PMD

A linear, real-time-temperature gradient of 63-71°C for 90 min was selected for mutation analysis of five known exon 3 variants of the LDLR gene. As shown in Figure 4.2, the mutations examined were W66G, E80K, D69G, delG303 and S78X. Normal control DNA was also loaded along-side the mutant controls. Gels were cast, and runs were set up as described in section 2.5.3. Gels were stained with ethidium and placed on a standard UV transilluminator for visualisation and photography. Dye migration was measured, as was the mobility of normal (unresolved) DNA.

Mutation W66G D69G S78X E80K DelG80 Normal sequence Amino-acid change (or nucleotide) Trp66Gly Asp69Gly Ser78Stop GluSOLys 303delG Base change TGG->GGG G A T ^G G T T Ç A ^ T A A GAG—>AAG G A G ^ G A

GC clamp + Forward primer FH195 G C T C G G C C T C A G T G G G T C T T T C C T T T G A G T G A C A G T T C A A T C C T G T C T C T T C T G T A G T G T C G A G C C G G A G T C A C C C A G A A A G G A A A C T C A C T G T C A A G T T A G G A C A G A G A A G A C A T C A C A C T G T C A C C T G C A A A T C C G G G G A C T T C A G C T G T G G G G G C C G T G T C A A C C G C T G C A T T C C T C G A C A G T G G A C G T T T A G G C C C C T G A A G T C G A C A C C C C C G G C A C A G T T G G C G A C G T A A G G A G V T C K S G D F S C G G 5 0 R V N R C 60 2 3 4 5 7 8 9 * * * * * * * A G T T C T G G A G G T G C G A T G G C C A A G T G G A C T G C G A C A A C G G C T C A G A C G A G C A A G G C T G T C T C A A G A C C T C C A C G C T A C C G G T T C A C C T G A C G C T G T T G C C G A G T C T G C T C G T T C C G A C A G F W R C D G Q V D C D N G S D E Q G C 6 6 6 9 7 0 8 0 8 1 •k # 1 3 G T A A G T G T G G C C C T G C C T T T G C T A T T G A G C C T A T C T G A G T C C T G G G G A G T G G T C T G A C T T C A T T C A C A C C G G G A C G G A A A C G A T A A C T C G G A T A G A C T C A G G A C C C C T C A C C A G A C T G A A FH 196 Reverse primer

Figure 4.2. Amplified sequence and mutations of exon 3 of the LDLR gene. * indicates presence of the mutation; = = = == is position of the primers. Amino acid and codon numbers are shown below complementary strand. Mutations: 1= delG235; 2= W66G; 3= D69G; 4= S78X; 5= E80K; 6= E80X; 7= delG303; 8= Q81X; 9= delCTGT309. (Adapted from FH database, http://www.ucl.ac.uk/fh). Mutations E80X, Q81X, delCTGT309 and delG235 were not used in the development of PMD but were identified in a sample of heterozygous FH patients (section 4.7.5).