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Letters |
1
Laboratory of Atherosclerosis Genetics, Department of Clinical Chemistry, Centre for Laboratory Medicine, Tampere University Hospital, and, University of Tampere, Medical School, FIN-33521 Tampere, Finland
2
Department of Medicine, Helsinki University Central Hospital, FIN-00029 Helsinki, Finland
a Address correspondence to this author at: Tampere University Hospital, Centre for Laboratory Medicine, Department of Clinical Chemistry, Laboratory of Atherosclerosis Genetics, FinnMedi 2, 3rd Floor, PO Box 2000, FIN-33521 Tampere, Finland. Fax 358-3-2474168; e-mail riikka.malin{at}cedar.csc.fi
To the Editor:
The angiotensinogen (AGT) gene encodes the precursor of the vasoactive hormone angiotensin II, which is the effector peptide of the renin-angiotensin system. A polymorphism of the AGT gene, consisting of two alleles coding for methionine (M) or threonine (T) at position 235, has been associated with essential hypertension (1), diabetic nephropathy (2), and coronary heart disease (3). Currently available but time-consuming methods for M/T235 genotyping include PCR amplification followed by denaturing gradient gel electrophoresis, sequencing, minisequencing (4), or restriction endonuclease digestion. Here we present a method based on the single-step LightCycler technology that uses a rapid PCR amplification followed by analysis of the melting behavior of fluorophore-labeled hybridization probes (5). In genotyping with two labeled probes, the shorter detection probe covers the polymorphic site and melts off the template at a lower temperature than the longer anchor probe. In the presence of mutation, a mismatch between the detection probe and the amplified sequence lowers the melting temperature of the detection probe.
Genomic DNA from patients was extracted from blood according to standard procedures. To confirm the LightCycler genotyping results, PCR products were analyzed by solid-phase minisequencing (4) and by sequencing using an ABI Prism 377 automated sequencer (PE Applied Biosystem). Six DNA samples, two of each AGT genotype, were randomly selected. As reaction buffer in the PCR, the LightCycler DNA Master Hybridization Probes 10x buffer (Roche Molecular Biochemicals) with a final Mg2+ concentration of 4 mmol/L was used. Unspecific primer elongation before PCR was prevented by incubating the buffer with TaqStart Antibody (Clontech Laboratories) according to the manufacturer’s instructions. PCR was performed in a reaction volume of 20 µL with 0.3 µmol/L of each primer (5'-CTCTATCTGGGAGCCTTG-3' and 5'-GTTTGCCTTACCTTGGAA-3') and 0.2 µmol/L of the anchor and detection probes (GENSET SA). The detection probe 5'-CCCTGATGGGAGCCAGTG-3' was labeled at the 3' end with fluorescein; the anchor probe 5'-GACAGCACCCTGGCTTTCAACAC-3' was labeled with LightCycler Red 640 at its 5' end and modified at the 3' end by phosphorylation to block extension.
The PCR was performed in a LightCycler instrument and included initial denaturation at 94 °C for 45 s, followed by 50 cycles of denaturation (94 °C for 0 s, with a temperature transition rate of 20 °C/s), annealing (57 °C for 5 s, 20 °C/s), and extension (72 °C for 20 s, 3 °C/s). After amplification, we recorded the melting curve by cooling the reaction mixture to 50 °C for 3 min, and then by slowly raising the temperature to 85 °C at 0.2 °C/s. The fluorescence signal (F) was continuously monitored during the temperature ramp and then plotted against temperature (T) to obtain melting curves for the samples (F vs T). The melting curves were subsequently converted to derivative melting curves [-(dF/dT) vs T].
The derivative melting curves of the MM, MT, and
TT genotypes of the AGT gene are shown in Fig. 1
. The genotypes of the six specimens matched the genotypes
obtained from sequencing and minisequencing. The melting peak of
samples homozygous for the M allele was at 63 °C, whereas in samples
homozygous for the T allele, the melting peak was at 54 °C. The
heterozygous samples contained both M and T alleles and produced both
peaks. The method can genotype 32 samples in <45 min with a single
sample processing step.
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We conclude that real-time PCR with fluorescence probe melting curves is an attractive alternative to labor-intensive methods for detection of AGT M/T235 polymorphisms.
Acknowledgments
The study was supported by the Medical Research Fund of the Tampere University Hospital and the Finnish Foundation for Cardiovascular Research.
References
The following articles in journals at HighWire Press have cited this article:
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  H. Sanada, J. Yatabe, S. Midorikawa, S. Hashimoto, T. Watanabe, J. H. Moore, M. D. Ritchie, S. M. Williams, J. C. Pezzullo, M. Sasaki, et al. Single-Nucleotide Polymorphisms for Diagnosis of Salt-Sensitive Hypertension Clin. Chem., March 1, 2006; 52(3): 352 - 360. [Abstract] [Full Text] [PDF]
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