The insertion-deletion (I/D) polymorphism at the ACE locus was typed using a previously described PCR-based method (Rigat et al. 1992). Nine polymorphisms, T-5991C, A-5466C, T-3892C, A-240T, T-93C, T1237C, G2215A, G2350A and 4656(CT)3/2, that were originally described by Villard and colleagues (1996), were genotyped following PCR amplification (see table 4 in the Keavney et al. paper).

The 4656(CT)3/2 polymorphism was PCR amplified using 50 ng of genomic DNA, 0.9 nmol of each dNTP, 9 pmol of each primer and 0.4 U of AmpliTaqä in a total vol. of 15 m l 1´ (NH4)2SO4 buffer with 2 mM MgCl2. An initial denaturation step of 94 ° C for 1.5 min was followed by 35 cycles of: 95 ° C for 20 s, 60 ° C for 30 s, and 72 ° C for 10 s. The forward primer was labelled with a fluorescent tag (FAM). Products were separated by electrophoresis on a denaturing polyacrylamide gel on a 373A Sequencer (Applied Biosystems) and analysed as described previously (McKenzie et al. 1995).

The T-93C polymorphism was amplified using 100 ng of DNA, 3 nmol of each dNTP, 24.8 pmol of each primer and 4 U of AmpliTaqä in a volume of 50 m l of 1´ (NH4)2SO4 buffer with 2 mM MgCl2 and 10% v/v glycerol. An initial denaturation step of 94 ° C for 1.5 min was followed by 10 cycles of: 96 ° C for 45 s, 65 ° C for 30 s with a decrement of 0.5 ° C each cycle (i.e. a "touchdown") and 72 ° C for 15 s, then 30 cycles of: 95 ° C for 30 s, 60 ° C for 30 s, and 72 ° C for 15 s.

The A-240T polymorphism was amplified using 3 m l of a 1/25 dilution of the T-93 C PCR product, 1.5 nmol of each dNTP, 12.5 pmol of each of the appropriate oligonucleotide primers and 0.75 U of AmpliTaqä in a total volume of 25 m l 1X (NH4)2SO4 buffer with 2 mM MgCl2. An initial denaturation step of 94 ° C for 1.5 min was followed by 20-35 cycles of: 95 ° C for 20 s, 60 ° C for 30 s and 72 ° C for 10 s.

The reaction mix for the other polymorphisms (T-5991C, A-5466C, T-3892C, T1237C, G2215A and G2350A) was the same as for A-240T with the exception that 100 ng of genomic DNA was used. An initial denaturation step of 94 ° C for 1.5 min was followed by 35 cycles of: 95 ° C for 20 s, 55-60 ° C for 30 s and 72 ° C for 10 s. The annealing temperatures and optimal MgCl2 concentrations were determined by titration (table 4).

The T-93C and G2215A polymorphisms alter recognition sequences for restriction endonucleases thus providing a straightforward method for detection. For the other polymorphisms, a "half-site" for an enzyme was created (or destroyed). By introducing a mismatch in the primers used to amplify across the polymorphism, the recognition site could be "completed". Restriction digests were performed using 5-10 m l of the PCR product with 2-5 U of the appropriate enzyme in a volume of 20-25 m l. Reaction buffers and temperatures were those recommended by the manufacturer. Table 4 shows the restriction enzymes that were used (New England Biolabs). Fragments were separated on ethidium-stained agarose gels and visualised on a UV trans-illuminator.

If you wish to enquire further with respect to the experimental conditions then please contact Dr. Colin McKenzie, Tropical Metabolism Research Unit, University of the West Indies, Mona, Kingston 7, Jamaica.  His e-mail address is colin.mckenzie02@uwimona.edu.jm

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Last edited 20th August 1998