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Genotyping of the angiotensin I-converting enzyme gene insertion/deletion polymorphism by the TaqMan method.

Intron 16 of the angiotensin I-converting enzyme gene (ACE) contains an insertion/ deletion (I/D) polymorphism that is characterized by the presence (I allele) or absence (D allele) of a 289-bp incomplete alu type repeat sequence (1,2). The D allele has been related to higher concentrations of ACE mRNA in cells and increased ACE concentration and activity in plasma and serum (1,3-6). There is great continuing interest in the link between the ACE I/D polymorphism and interindividual variations in physiologic properties and disease susceptibility. Reported associations include physical activity and endurance, drug response, and neuropathologic, cardiac, and cardiovascular diseases (7-20). However, a considerable number of findings are in disagreement with the existence of such relationships (21-31), and more work is required before the role of the ACE I/D polymorphism in health and disease can be firmly established.

Conventional genotyping of the ACE I/D polymorphism involves PCR, using primers that flank the insertion sequence, and exploits the different migration velocities of I- and D-allele-specific PCR products during electrophoresis in gel matrices (32). Here we describe an assay for genotyping of the ACE I/D polymorphism that is based on the TagMan technique (33). The TaqMan method involves 2 allele-specific fluorogenic oligonucleotide probes and combines DNA amplification and genotype detection in a single assay. Usually, TagMan genotyping assays are designed and used for the analysis of single-nucleotide polymorphisms or small insertions/deletions (34-36). The new TagMan assay for the ACE I/D polymorphism extends the scope of the TagMan genotyping technique to the analysis of a polymorphism that is distinguished by the presence or absence of a relatively large sequence block. Special to this assay is the requirement of 3 different PCR primers to obtain the 2 types of allele-specific PCR products, instead of the 2 primers used in conventional TagMan assays. The binding sites of primers and probes on the I- and D-allele-specific DNA templates are shown in Fig. 1A. Primer pair AC111/ ACE112 gave rise to a D-allele-specific PCR product of 78 bp, and primer pair AC112/ACE113 gave rise to an I-allele-specific PCR product of 71 bp. The 367-by-long I-allele-specific amplicon produced by primer pair AC111 /ACE112 was not detectably multiplied in TagMan reactions that included DNA of genotype II or ID, as indicated by sample analysis using electrophoresis in a polyacrylamide gel and subsequent treatment of the gel with ethidium bromide solution. Allele-specific signaling of the probes was accomplished by the reporter dyes VIC (proprietary dye of Applied Biosystems) and 6-carboxyfluorescein (FAM), which were attached to the 5' ends of the I- and D-allele-specific oligonucleotides, respectively. Minor groove binder (MGB) groups were conjugated with the 3' ends of the oligonucleotides to facilitate formation of stable duplexes between the probes and their singlestranded DNA targets (37). Primers and probes were synthesized by Applied Biosystems and had the following structures:

Primer ACE111: 5'-CCCATCCTTTCTCCCATTTCTC-3' Primer ACE112: 5'-AGCTGGAATAAAATT000GAAAC-3' Primer ACE113: 5'-CCTCCCAAAGTGCTGGGATTA-3' I-Allele-specific probe (VIC-ACE100): VIC-5'-A000GTGATACAGTCA-3'-MGB D-Allele-specific probe (FAM-ACE100): FAM-5'-TGCTGCCTATACAGTCA-3'-MGB

[FIGURE 1 OMITTED]

Genomic DNA was extracted from peripheral blood leukocytes by use of the NucleoSpin Blood Quick Pure reagents (Macherey-Nagel). Reactions were carried out on 96-well microtiter plates. The assay volume was 22 [micro]L, which contained 11 [micro]L of the Absolute QPCR ROX Mix (ABgene); 150 nM each of the primers ACE111, ACE112, and ACE113; 150 nM I-allele-specific probe VIC-ACE100, 75 nM D-allele-specific probe FAM-ACE100, and 10-50 ng of DNA. The 2-step thermocycling procedure consisted of 35 cycles of denaturation at 92[degrees]C for 15 s and primer annealing and extension at 57[degrees]C for 1 min. After cycling on a GeneAmp PCR System 9600 or 9700 (Applied Biosystems), genotype calling was carried out on the ABI Prism 7000 Sequence Detection System (Applied Biosystems). A typical result is shown in Fig. 1B, which demonstrates the capability of the new TagMan assay to differentiate between the ACE I/D genotypes. We verified the ability of the TagMan system to provide correct genotype data by separate analysis of a limited number of samples (n = 150), using DNA sequencing or an established PCR genotyping system for the ACE I/D polymorphism, as described previously (30,38).

Using the new method, we determined the ACE I/D genotype in 1500 individuals of Caucasian origin who were included in a study designed to examine a possible association of the ACE I/D polymorphism with adverse events commonly occurring after interventions in coronary arteries. Written informed consent was obtained from all participating patients. The study protocol was approved by the institutional ethics committee, and the investigations were in accordance with the principles of the current version of the Declaration of Helsinki (39). In this series of patients, 299 (19.9%) were homozygous II, 737 (49.1%) were heterozygous ID, and 464 (30.9%) were homozygous DD. The observed ACE I/D genotype distribution was in Hardy-Weinberg equilibrium (P = 0.84) and in accordance with results obtained in other Caucasian populations (4,5,9,15,19,20,24,26,28,31).

The widely used conventional technique for ACE I/D genotyping, a combination of PCR and gel electrophoresis, is prone to misclassification of ID heterozygotes as DD homozygotes because of preferential amplification of the smaller D-allele-related sequence (28,40,41). To take into consideration the possibility of mistyping, it has been recommended samples typed as DD be subjected to a second, independent PCR including a primer pair that permits amplification only in the presence of the I allele but not the D allele (28,40,41). With the conventional method, accurate genotyping requires several time-consuming reaction steps, repeated transfer of material, and manual data acquisition.

The new TagMan genotyping system for the ACE I/D polymorphism is relatively simple to use and requires little hands-on time because it is a single-tube assay and allows for automated reaction setup, genotype determination, and data processing. Because post-PCR sample handling is not involved, the chance of sample mix-up or the possibility of carryover contamination is greatly reduced. The microtiter plate format and the option to process multiple plates in parallel make this method particularly suitable for approaches demanding high-throughput genotyping, such as clinical association studies involving large numbers of individuals. In addition, the TagMan system for the ACE I/D polymorphism may serve as a prototype for genotyping assays to be designed for other polymorphisms that are characterized by large insertions/ deletions.

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DOI : 10.1373/clinchem.2005.051656

Werner Koch, * Wolfgang Latz, Marianne Eichinger, Claudia Ganser, Albert Schomig, and Adnan Kastrati

(Deutsches Herzzentrum Munchen and 1. Medizinische Klinik, Klinikum rechts der Isar, Technische Universitat Munchen, Munich, Germany; * address correspondence to this author at: Deutsches Herzzentrum Munchen, Lazarettstrasse 36, D-80636 Munich, Germany; fax 49-89-1218-3053, e-mail wkoch@dhm.mhn.de)
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Title Annotation:Technical Briefs
Author:Koch, Werner; Latz, Wolfgang; Eichinger, Marianne; Ganser, Claudia; Schomig, Albert; Kastrati, Adnan
Publication:Clinical Chemistry
Date:Aug 1, 2005
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