Determination of Angiotensin-Converting Enzyme Gene Polymorphisms: Stepdown PCR Increases Detection of Heterozygotes

The angiotensin-converting enzyme (ACE) gene product plays an important role in cardiovascular homeostasis. An insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene, with insertion polymorphism containing three more Alu-repeat sequences, was reported to be a determining factor of the plasma ACE concentration, and the D polymorphism has been found to be associated with certain cardiovascular diseases (1)(2)(3)(4)(5). Controversy exists, however, regarding the strength of the association. The diversity of conclusions has been attributed to methodological and technical variations in detection of the polymorphisms (6)(7). The preferential amplification of the D allele of the ACE gene by the PCR reported by Rigat et al. (8) was thought to be one cause. This PCR method occasionally mistyped ID heterozygotes as DD homozygotes (9). The probability of this mistyping has been estimated to be ∼5–10% (6)(7). A confirmatory PCR method, which requires an additional third PCR primer inside the Alu sequence of the I allele, was proposed to minimize the mistyping of the I allele as a D allele (9). Although this PCR technique was reported to be 100% in the typing of ACE gene polymorphisms, problems with the preferential amplification of multiplexed PCR are not entirely excluded with this method (10)(11). A stepdown PCR method, modified from touchdown PCR, has recently been used in several molecular studies (12). This method involves initial PCR annealing temperatures higher than the melting point of the primers, followed by annealing temperatures reduced stepwise to the melting point. This method should result in higher amplification specificity and greater yield. We compared the stepdown PCR with the conventional method from Rigat et al. (8) and with confirmatory PCR in the amplification efficiency of the ACE gene. We collected blood samples from …