Single Strand Conformation Polymorphism (SSC?)

2.4.4.1 Minimum use of radioisotope a-^^P

a-^^P deoxycytosine triphosphate (a-^^P dCTP) was used to label the PCR products. To minimise the exposure to radioactivity, a PCR polymerase mix containing one tenth (1/10) the usual concentration of dCTP was used (0.02 mM dCTP, 0.2 mM dATP, 0.2 mM dGTP and 0.2 mM dTTP) together with a ten-fold reduction o f neat a-^^P dCTP (t] / 2 =14 days). Therefore, 2 pi of hot a-^^P dCTP at 250 pCi/pL at 9.25 MBq

2 .4 .4 2 S a m p le p rep a ra tio n fo r S S C P a n a ly sis

For the detection of genetic variability within GTP cyclohydrolase 1 gene (GCH l) by SSCP, the promoter region was amplified by using five overlapping primer pairs. PCR was performed in five microtitre plates as described in Section 2.4.1 with few modifications. The protocol adopted for each amplification was as follows:

1. Two pi of 44 DNA samples from Master Array 1 (NPHSII) was dispensed in a 96- well plate (Hybaid Omnigene) and dried (75°C / 10 min).

2. 1500 pi PCR cocktail was prepared for 50 samples, that is enough for half a microtitre plate, i.e. 44 samples, 4 blanks (negative controls) and 2 extras. See Table 5.2 {Section 5.3.3, Chapter 5) for PCR cocktail preparation.

3. 2 pi o f a-^^P dCTP was added to 1.5 ml (30 pi x 50 samples) o f cocktail mix and the radioactive mix was distributed into the blank and dried DNA templates wells. The cocktails were overlaid with 20 pi paraffin oil.

4. The amplification cycles were 95°C for 5 min followed by 35 cycles o f three consecutive steps each lasting 45 sec: dénaturation at 95°C, annealing step with a Ta optimised for each primer set {Table 5.1, Section 5.3.2, Chapter 5) and extension at 72°C. The same program was used for both radioactive and non­ radioactive PCRs.

2.4.4 3 SSCP double gel set-up

For convenience and high sample throughput, samples were run on two gels simultaneously on a vertical electrophoresis apparatus. The gels, prepared as described in Section 2.4.3.4, were poured between three glass plates. When clamped, the arrangement of the plates was a large plate, which was the 'lowest' and outermost one and two smaller plates, which were 'middle' and 'uppermost'. The glass plates were scrubbed with mercury chloride and cleaned with detergent (Fairy Liquid Excel, Proctor & Gamble Ltd) followed by 100% ethanol and distilled water. Repelcote (BDH, Poole, UK) was applied thinly to the four glass faces which make contact with the two gels.

A pair o f spacers (0.4 x 10 x 400 mm) were placed on top of the silanized side o f the large 'lowest' plate (33 x 42 cm). The small plate (33 x 39 cm) which had both sides repelcoted was put on top of the spacers and a second set o f spacers were arranged on top o f this 'middle' plate. The silanized side o f the 'uppermost' small plate (33 cm x 39 cm) was placed on top of this arrangement. The sides of the three plates were aligned and clamped with bulldog clips. The bottom gel was injected on the large plate by using a syringe, whilst tapping gently at the gel front so as to avoid any bubbles. The flat side of a shark comb was inserted and the gel allowed to set for 10-15 min. The second gel was then poured in a similar manner and another comb inserted. To ensure that both gels were properly set and with no leakage, the gels were allowed to set for 2- 3 hours.

2.4 4.4 S S C P g el elec tro p h o resis

The amplified PCR samples were subject to SSCP on 7.5% polyacrylamide non­ denaturing gels. The procedures were the same for all the five gels. Three pi of radioactive PCR mix of all samples were transferred into a 96-well plate containing 6 pi o f SSCP loading dye in separate wells. The mixture was mixed gently with the pipette. The samples were denatured (95°C / 5 min) and chilled immediately on wet ice. The top buffer tank of the electrophoresis apparatus was filled with Ix TBE and after ensuring that the top tank had no leakage, the bottom tank was filled with Ix TBE. The two combs were removed, rinsed and the teeth of both combs were inserted gently in both gels and the samples loaded in the wells with the multi pipette.

Electrophoresis conditions were 300 V at 21-25°C for about 21 hours and after that period the electrophoresis apparatus was stopped and the top buffer tank emptied. The plates were removed and prized apart. The gels, which remained stuck to one o f the plates, were transferred to Whatman 3 mm chromatographic paper and dried (80°C / 30 min). They were exposed to a Hyperfilm MP (Amersham, UK) for 2 days at -70°C with an intensifying screen in a film cassette. The film was developed by dipping it respectively in silver nitrate solution (which deposits on the DNA bands), in diluted acetic acid (to stop the reaction) and finally in a solution o f formaldehyde and sodium carbonate (which reduces the silver). The film was washed with tap water and dried.

2.4.4 5 Reading an SSCP profile

The X-ray films were analysed for SSCP variants, which have a different pattern of migration on the polyacrylamide gel due to conformational changes. A few PCR amplified samples, exhibiting the three types o f mobility shifts on the SSCP polyacrylamide gel, were purified and sequenced {Section 5.3.6, Chapter 5).

2.4 4.6 Template preparation for DNA sequencing

Prior to sequencing, the QIAquick Gel Extraction Kit (QIAGEN) was used to extract PCR fragments for direct use as templates during sequencing. The main steps were solubilisation of the gel slice, binding of the double-stranded DNA to a silica membrane, washing of all contaminants (i.e. salts, excess primers, agarose, dyes, ethidium bromide, oils, unincorporated nucleotides...) and elution of the pure bound DNA with ddH20.

2.4.4.7 Cycle sequencing of purified DNA fragments

Both the forward and reverse strands of the purified DNA fragments were used as templates for further amplification by cycle sequencing using the primers from Section 2.4.4.2. A commercial kit, the ABI PRISM™ dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems), was used and for each sequencing reaction, the cocktail was prepared according to the manufacturer's

Briefly, about 60 ng (~ 1 |o,l) of PCR template (amplified and purified as described in Sections 2AA .2 and 2.4.4.6), 3.2 pmole (3.2 pi) o f forward or reverse primer, 8 pi of

pre-mix (buffer, enzyme, dNTPs and dye-labeled ddNTPs) and 7.8 pi ddH%0 were added to labelled sequencing tubes. The reactions were overlaid with 20 pi mineral oil. This amplification process employs only a single primer during the repeated cycles of thermal dénaturation, primer annealing and polymerisation within each sequencing tube.

The plate was placed in the thermocycler (GeneAmp PCR 9700, PE Applied Biosystems) with the following cycle parameters:

Step 1: 96°C for 10 sec (i.e. dénaturation step) Step 2: 50°C for 5 sec (i.e. annealing step) Step 3: 60°C for 4 min (i.e. extension step)

Step 4: Repeat Steps 1, 2 and 3 sequentially for 25 cycles