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Technical Briefs |
1
Central Laboratory and
2
Department of Neurology and Neurophysiology, Pándy Kálmán County Hospital, H-5700 Gyula, Semmelweis 1, Hungary;
aaddress correspondence to this author at: Central Laboratory, Pándy Kálmán County Hospital, H-5700 Gyula, Budapest krt. 58, Hungary; fax 36-66-463474, e-mail fsoma99{at}hotmail.com
The serum angiotensin-converting enzyme (ACE) concentration depends on the ACE gene insertion/deletion (I/D) polymorphism. This insertion is a 287-bp-long alu repetitive sequence localized in intron 16 of the gene (1). The D/D genotype is associated with a higher serum ACE concentration, and it has been demonstrated that it may be associated with ischemic heart diseases (2)(3)(4). We are interested in the role of the ACE D allele in ischemic stroke (5)(6).
The PCR method reported by Rigat et al. (1) has been modified several times because of preferential amplification of the D allele. In the past, I/D heterozygotes had been mistyped as D/D homozygotes; the extent of this misclassification has been estimated at 
5% (2)(7)(8).
Some of the modified techniques are listed below.
The confirmatory PCR method proposed by Shangmugam et al. (9) uses a third PCR primer inside the I allele, just like multiplexed PCR. Other authors consider that multiplexed PCR is not acceptable in this method (10).
The step-down PCR described by Chiang et al. (11) is a modified touchdown PCR involving some initial cycles with an annealing temperature higher than the melting point of the primers, followed by annealing temperatures reduced stepwise to the melting point. Unfortunately, this method is time-consuming, and some thermocyclers are not programmable for this kind of method.
A better solution, which uses a second, independent PCR amplification with a primer pair that recognizes an insertion-specific sequence, has been described by Lindpainter et al. (2). In this method, the I/D genotypes that were mistyped as D/D in the first PCR reaction can be detected. Unfortunately, this method is also time-consuming.
To reduce the time requirements, we suggest a rapid PCR with fluorescently labeled oligonucleotide hybridization probes on the LightCyclerTM instrument (Roche Diagnostics) with subsequent fluorescent probe melting point analysis. The proposed assay has been confirmed by acrylamide gel electrophoresis.
The fluorescence hybridization probe was constructed to fit into the insertion-specific sequence, which can also be amplified by the confirmatory primer pair mentioned above (2). Thus, validation of the D/D genotype with the independent PCR reaction is feasible with the same fluorescent probes.
Previously, hybridization probes have been constructed to detect point mutations or short insertions. In the case of short insertions, the hybridization probe bridges the whole mutation site (12)(13). For the detection of a long insertion, a general solution could involve designing the probe to overlap 1–5 bases on the insertion. The scheme of our solution is shown in Fig. 1A
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Genomic DNA was extracted from 200 µL of peripheral blood anticoagulated with EDTA with the QIAamp blood reagent set (Qiagen) according to the manufacturer’s instruction. All blood samples were kept at -20 °C until DNA isolation.
PCR was performed in disposable capillaries (Roche Diagnostics). The reaction volume was 10 µL, containing 1 µL of DNA (40–80 ng), 0.2 µM each of the primers reported by Rigat et al. (1), 1 µL of reaction buffer (LightCycler DNA master hybridization probes 10x buffer; Roche Diagnostics), 0.4 µL of 25 mM MgCl2 stock solution, 0.5 µL of dimethyl sulfoxide, and 0.1 µM each of the probes. The detection probe specific for the 3' end of the insertion (underlined four bases in the 5' end of the probe) was labeled at the 3' end with fluorescein (5'-CGT GAT ACA GTC ACT TTT ATG-3'). The anchor probe (5'-GGT TTC GCC AAT TTT ATT CCA GCT CTG-3') was labeled with LightCycler Red 640 at the 5' end and was modified at the 3' end by phosphorylation to block extension.
The PCR conditions were as follows: initial denaturation at 95 °C for 60 s, followed by 40 cycles of denaturation (95 °C for 0 s, 20 °C/s), annealing (61 °C for 10 s, 20 °C/s), and extension (72 °C for 15 s, 20 °C/s). The melting curve analysis consisted of 1 cycle at 95 °C for 10 s and 40 °C for 10 s, followed by an increase of the temperature to 65 °C at 0.2 °C/s. The fluorescence signal (F) was monitored continuously during the temperature ramp and then plotted against the temperature (T). These curves were transformed to derivative melting curves [(-dF/dT) vs T]. The derivative melting curves for the three genotypes (I/I, I/D and D/D) are depicted in Fig. 1B
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The PCR conditions were the same when the confirmatory primer pair were used, except that the annealing temperature was 67 °C. In this case, the derivative melting curve exhibited only the peak characteristic of the insertion allele.
Of 103 patient samples tested, 23.3% were I/I, 44.7% were I/D, and 33.0% were D/D.
The proposed technique and the electrophoresis yielded identical results. No ACE D/D misclassification was found even when the confirmatory, independent primer pair was used.
References
The following articles in journals at HighWire Press have cited this article:
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  E. M. W. van de Garde, H. Endeman, V. H. M. Deneer, D. H. Biesma, F. A. Sayed-Tabatabaei, H. J. T. Ruven, H. G. M. Leufkens, and J. M. M. van den Bosch Angiotensin-Converting Enzyme Insertion/Deletion Polymorphism and Risk and Outcome of Pneumonia Chest, January 1, 2008; 133(1): 220 - 225. [Abstract] [Full Text] [PDF]
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 Z Szolnoki, F Somogyvari, A Kondacs, M Szabo, L Fodor, J Bene, and B Melegh Evaluation of the modifying effects of unfavourable genotypes on classical clinical risk factors for ischaemic stroke J. Neurol. Neurosurg. Psychiatry, December 1, 2003; 74(12): 1615 - 1620. [Abstract] [Full Text] [PDF]
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