Rapid Detection of Angiotensinogen M/T235 Polymorphism by Fluorescence Probe Melting Curves To the Editor: The angiotensinogen (AGT) gene en- codes the precursor of the vasoactive hormone angiotensin II, which is the effector peptide of the renin-angio- tensin system. A polymorphism of the AGT gene, consisting of two al- leles coding for methionine (M) or threonine (T) at position 235, has been associated with essential hyper- tension (1), diabetic nephropathy (2), and coronary heart disease (3). Cur- rently available but time-consuming methods for M/T235 genotyping in- clude PCR amplification followed by denaturing gradient gel electro- phoresis, sequencing, minisequenc- ing (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 melt- ing 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 se- quencing 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 10 buffer (Roche Molecular Biochemicals) with a final Mg 2+ 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 man- ufacturer’s instructions. PCR was performed in a reaction volume of 20 L with 0.3 mol/L of each primer (5' -CTCTATCTGGGAGCCTTG-3' and 5'-GTTTGCCTTACCTTGGAA- 3') and 0.2 mol/L of the anchor and detection probes (GENSET SA). The detection probe 5'-CCCTGATGG- GAGCCAGTG-3' was labeled at the 3' end with fluorescein; the anchor probe 5'-GACAGCACCCTGGCTT- TCAACAC-3' was labeled with LightCycler Red 640 at its 5' end and modified at the 3' end by phosphor- ylation 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 ampli- fication, 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 moni- tored 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 de- rivative 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 minisequenc- ing. The melting peak of samples homozygous for the M allele was at 63 °C, whereas in samples homozy- gous 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 pro- cessing step. We conclude that real-time PCR with fluorescence probe melting curves is an attractive alternative to labor-intensive methods for detec- tion of AGT M/T235 polymor- phisms. The study was supported by the Medical Research Fund of the Tam- pere University Hospital and the Finnish Foundation for Cardiovascu- lar Research. Fig. 1. Derivative melting curves of angiotensinogen MM, MT, and TT genotypes. Melting curves of MM and TT homozygotes, an MT heterozygote, and no-template control (water) were obtained with allele-specific fluorescent probes. After PCR amplification, the temperature was slowly increased from 50 °C to 85 °C with continuous fluorescence monitoring. Fluorescence data were converted to derivative melting curves by plotting the negative derivative of the fluorescence with respect to temperature [-(dF/dT)] against temperature (T). 880 Letters