Mistyping of the human angiotensin-converting enzyme gene polymorphism: frequency, causes and possible methods to avoid errors in typing.
A polymorphism of the gene encoding the human angiotensin I-converting enzyme (ACE), which is defined by an insertion/deletion polymorphism in intron 16, has been identified as a candidate genetic locus in the development of cardiovascular and renal disease. We have demonstrated that the accuracy of ACE genotyping is critically dependent on the strategy of the PCR used in typing. Of 1238 individuals genotyped by a standard method, 335 were typed as DD, 645 as DI and 258 as II. However, when DD individuals were retyped using modified methods (including either 5% dimethyl sulphoxide, or a 'hot start') 35 of the original 335 samples (10.5%) were retyped as DI. In approximately half of these mistyped samples, PCR amplification was assessed as inefficient by the absence of a third faint heteroduplex band in a control ID sample: when the assay was repeated without any modifications, the mistyped samples were correctly genotyped. In the remainder, mistyping persisted. In these cases, the use of a third 'nested' PCR primer specific for the I allele was required for successful genotyping, providing a more reliable strategy without the need for further modification to the PCR technique. Our results suggest that the triple primer approach is the method of choice for accurate ACE genotyping.
Ueda
S
S
University Department of Medicine and Therapeutics, Western Infirmary, Glasgow, UK.
Heeley
R P
RP
Lees
K R
KR
Elliott
H L
HL
Connell
J M
JM
D004121:Dimethyl Sulfoxide; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D008297:Male; D007703:Peptidyl-Dipeptidase A / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
D007703:Peptidyl-Dipeptidase A; D004121:Dimethyl Sulfoxide