Ambiguities in angiotensin-converting enzyme allele genotyping: comparison of two methods

Angiotensin-converting enzyme (ACE) genotyping has been applied to many investigations of genetic risk factor analysis in hypertension and coronary heart disease. This study examined the reliability of the technique. Genotyping was conducted by the method of Rigat et al1. A 566bp segment of the ACE gene including the polymorphic 287bp insertion was amplified by PCR. PCR conditions optimised to our MJ Research PTC-96V thermal cycler were: 50ng of DNA (O.D. 260/280 > 1.8)/tube in 200μM dNTPs, 2mM MgCl2, 1× Taq reaction buffer (Promega Corp), 2.5U Taq polymerase at 94°C for 1 minute, 58°C for 1 minute and 72°C for 2 minutes. PCR samples were also run in 5% DMSO under the same conditions. Samples were electrophoresed on 1% agarose gels containing 0.01mg/mL ethidium bromide for 1.5 hours at 150V and alleles were typed under UV transillumination. Gels were run using native and denatured samples with/without DMSO in the PCR reaction. Significant differences were found between allele typings on the same PCR sample. It was possible to generate all genotypes in some cases by changing the conditions. Results were compared with those obtained by allele specific oligonucleotide (ASO) hybridisation of Southern blots from the agarose gels using probes to the D allele: (1431–51+1738–1758) and bp 1482–1532 of the insertion sequence (I allele). Mistyping rates of 42–15% depending on conditions employed were obtained with 136 samples compared to ASO typings. This implies that ACE genotypes need to be checked by ASO hybridisation to give reliable results. 1. Rigat B, Hubert C. Nucl Acid Res (1992) 20: 1433