PCR reactions were carried out in a volume of 25µl using 300ng of genomic DNA. The used primer pairs for delineating the polymorphism at position 1309 (Arg16→ Gly) were: 5’-CTT CTT GCT GGC ACC CAA TA-3’ (sense) and 5’-ACA ATC CAC ACC ATC AGA AT-3’ (antisense) or the same antisense primer and 5’-CTT CTT GCT GGC ACC CAA TG-3’ (sense). Use of these primers resulted in a product with a molecular size of 452 base pairs.
Allele-specific PCR was optimised by a standard magnesium titration (1.5, 2.5, 3.5 and 4.5 mM) and by raising the default annealing temperatures for increased specifity.
The polymorphism at position 1342 (Gln27→ Glu) introduced a C:C or G:G mismatch at the 3’ end of the sense oligonucleotide. Although the C:C mismatch reduces product yield by approximately 100 fold, the G:G mismatch appears not to affect amplification [164]. For this reason we introduced a second mismatch in both primers resulting in an additional G:C mismatch next to the allele specific mismatch. The used primers were: 5’-GGA CCA CGA CGT CAC GCA CC-3’ (sense) and 5’-ACA ATC CAC ACC ATC AGA AT-3’ (antisense) or the same antisense primer and 5’-GGA CCA CGA CGT CAC GCA CG-3’ (sense). Use of these primers resulted in a product with a molecular size of 419 base pairs.
One international unit of Dynazyme (Dynal) was used with reaction buffers supplied by the manufacturer, 200 µM of each deoxynucleotide triphosphate (Pharmacia) and 1.5 mmol/l magnesium chloride. Primers (Table 3.2) were used at a final concentration of 0.4 to 0.8 µM each. Amplification was over 30 cycles at 95℃C for 30 seconds, annealing temperature for 35 seconds, and 72℃C for 35 seconds. An initial denaturation step (95℃C for 1 minute) and a final extension step (72℃C for 3 minutes) was added. The annealing temperature for the 1309 fragment was at 68℃C, for the 1342 fragment at 65℃C. Amplification was performed in 200µl tubes in a compact silver microblock of an OmniGene thermal cycler (Hybaid Ltd., England) using tube control and an oil overlay.
Seven microlitres of the PCR reactions were then electrophoresed on 1.5% agarose gels and visualised with ethidium bromide staining and ultraviolet illumination. Each sample was analysed twice, and direct sequencing of the region containing the Arg16→ Gly variant was undertaken, using Thermus aquaticus fluorescent cycle sequencing (TaqFS sequencing kit, Perkin-Elmer) on an automated (ABI 377) sequencer (Applied Biosystems) to confirm the robustness of the genotypes of 18 individuals. For details on the used sequencing method, see section 4.2.5.
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© 2001 Alexander Binder