The polymerase chain reaction for all three fragments was done in a 25 
l
volume using 0.2 g of genomic DNA extracted from whole blood (see
section DNAprep). In addition to the sample DNA, the mixture contained
2.5 l tenfold Taq polymerase buffer (Promega), 1.5 mM MgCl 
,
200 M of each deoxynucleotide triphosphate (Pharmacia) and 1 unit
Taq polymerase (Promega). Primer (see table tab:RFLPprimers)
were used at a final concentration of 0.4 to 0.8 M each. For the third
fragment, the reaction volume was doubled (50 l) when polymorphism
analysis was performed on agarose gels.
The amplification was performed in 0.2 ml tube-strips in a compact silver microblock of an Omnigene thermal cycler (Hybaid Ltd., England). The block was equipped with a heated lid. This heating plate heats the air temperature at the top of each reaction mixture to a temperature that is permanently higher than the sample temperature (120psy176 C). This elevated air temperature relative to the sample temperature minimizes evaporation so that there is no condensation of the reaction mixture as the reaction mixture is repeatedly heated and cooled. This technique eliminates the necessity of an oil overlay. The sample temperatures were controlled based on a remote thermistor probe mounted in an appropriate tube in the block. Tube control means, the actual sample temperatures are held at the programmed temperature for the programmed time, whereas with block control, there will inevitably be a lag between the block reaching target temperature and the samples reaching target temperature. Thus the used incubation times have been reduced dramatically.
For cycle protocol, an initial denaturation step (95psy176 C for 1.5 minutes) was followed by an annealing step (35 s), extension step (72psy176 C) starting at one minute with an one second time increment, and denaturing step (94psy176 C) for 20 s. The annealing temperature was 48-57psy176 C. After 25 cycles, a final extension step (72psy176 C) for 5 minutes was added.