title: Angiotensin-converting enzyme insertion/deletion polymorphism genotyping error: the cause and a possible solution to the problem.
abstract: Rigat and colleagues were the first ones to develop a rapid PCR-based assay for identifying the angiotensin converting enzyme insertion/deletion (I/D) polymorphism. Due to a big difference between the length of the wild-type and mute alleles the PCR method is prone to mistyping because of preferential amplification of the D allele causing depicting I/D heterozygotes as D/D homozygotes. The aim of this study was to investigate whether this preferential amplification can be repressed by amplifying a longer DNA fragment in a so called Long PCR protocol. We also aimed to compare the results of genotyping using five different PCR protocols and to estimate the mistyping rate. The study included 200 samples which were genotyped using standard method used in our laboratory, a stepdown PCR, PCR protocol with the inclusion of 4 % DMSO, PCR with the use of insertion specific primers and new Long PCR method. The results of this study have shown that accurate ACE I/D polymorphism genotyping can be accomplished with the standard and the Long PCR method. Also, as of our results, accurate ACE I/D polymorphism genotyping can be accomplished regardless of the method used. Therefore, if the standard method is optimized more cautiously, accurate results can be obtained by this simple, inexpensive and rapid PCR protocol.
authors: University Department of Chemistry, University Hospital Centre "Sestre Milosrdnice", Vinogradska 29, 10 000, Zagreb, Croatia. andrea.saracevic@gmail.com
authors: Andrea
authors: A
authors: Saracevic
authors: Ana-Maria
authors: AM
authors: Simundic
authors: Ivana
authors: I
authors: Celap
authors: Valentina
authors: V
authors: Luzanic
mesh_terms: D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods / Q000592:standards*; D006801:Humans; D054643:INDEL Mutation; D007703:Peptidyl-Dipeptidase A / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results
chemical_list: D007703:Peptidyl-Dipeptidase A
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