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Letters |
1
Diagnostic Laboratory, Becker, Olgemöller und Kollegen, Führichstrasse 70, 81671 Munich, Germany
2
Roche Molecular Biochemicals, Nonnenwald 2, 82372 Penzberg, Germany
aAuthor for correspondence. Fax 49-89-450917-300; e-mail burggraf{at}labor-bo.de.
To the Editor:
A common methionine/valine polymorphism at codon 129 of the prion protein gene modulates disease susceptibility and phenotypic variability of human prion diseases (1)(2). All patients with the new variant of Creutzfeldt-Jakob disease (vCJD) were homozygous for methionine at codon 129 (3). We developed a genotyping assay that uses rapid-cycle PCR and melting point analysis of fluorogenic hybridization probes, but we encountered an unexplained artifact of melting behavior that may lead to misinterpretation when using this method. Asymmetric PCR allowed reliable genotyping results.
Primers (5'-CACAGTCAGTGGAACAAG-3' and 5'-GTACACTTGGTTGGGGT-3'; GenBank accession no. HSU29185, positions 25735–25752 and 25935–25919, respectively) and probes (anchor probe, 5'-CCGAAATGTATGATGGGCCTGCTCAT-fluorescein-3'; detection probe, 5'-LC-Red640-CACTTCCCAGCACGTAGCC-phosphate-3'; GenBank accession no. HSU29185, positions 25874–25849 and 25846–25828, respectively) were designed using a beta version of the LightCycler probe design software (Ver. 0.99.11; Roche Molecular Biochemicals). This software uses recommended methods for probe design (4) and calculates melting temperatures (Tms) by a nearest-neighbor method. PCR reactions were carried out in a final volume of 10 µL in glass capillaries (Roche Molecular Biochemicals). Each reaction mixture contained 5 pmol of forward primer, different amounts of reverse primer (0.5–5 pmol), 2 pmol each of anchor and detection probe, 3 mmol/L MgCl2, 1 µL of LightCycler-FastStart DNA Master Hybridization Probes (which includes reaction buffer, nucleotides, and Taq polymerase), and 2.5 µL of genomic DNA. The thermocycling conditions were as follows: 95 °C for 10 min for initial denaturation and activation of Taq polymerase and 45 cycles of 95 °C for 0 s, 55 °C for 5 s, and 72 °C for 10 s, with a ramping rate of 20 °C/s. Melting analysis was performed by heating the capillary at 95 °C for 10 s, followed by incubation at 45 °C for 1 min and then slow (0.1 °C/s) heating to 80 °C.
The results of the melting point analysis were unexpected. Both a methionine/valine heterozygous sample and a valine homozygous sample lacked a valine peak, which was expected to occur at 
65 °C (i.e., the calculated Tm for the detection probe without mismatch).
To evaluate the quality of the hybridization probes, we used single-stranded oligonucleotides complementary to the probe set in a melting point experiment without prior PCR amplification. Both so-called complements, one corresponding to the valine genotype (no mismatch) and the other corresponding to the methionine genotype (one mismatch), showed the calculated melting peaks of 65 and 58 °C, respectively (Fig. 1A
).
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Because the probes had now been shown to work well on a single-stranded template, an asymmetric PCR system was devised to increase the amount of single-stranded PCR product available as target for the probes. In this system, the target DNA was amplified in the presence of a fixed amount of forward primer and various dilutions of reverse primer. The heterozygous (methionine/valine) sample could be successfully amplified with all concentrations of the reverse primer, from 5 pmol (equal to forward primer) to only 0.5 pmol (Fig. 1B
). However, the reaction containing equal amounts of primers (Fig. 1C
, curve 1), revealed only one peak at 58 °C in the melting point analysis. The correct result for the heterozygous sample, with the two expected peaks (at 58 and 68 °C), was obtained only when the concentration of the reverse primer was 1/5 to 1/10 that of the forward primer (Fig. 1C
, curves 3 and 4). In the presence of 2.5 pmol of reverse primer, the valine peak at 68 °C was present but was substantially smaller than the methionine peak at 58 °C (Fig. 1C
, curve 2).
To check the influence of the probe composition, we designed a second set of hybridization probes (anchor probe, 5'-LC-Red640-GCCCCCCACCACTGCCC-phosphate-3'; detection probe, 5'-AGCACGTAGCCGCCA-fluorescein-3'; GenBank accession no. HSU29185; positions 25821–25805 and 25838–25824, respectively). This alternative probe system produced identical results in the melting point analysis of the heterozygous sample, i.e., only asymmetric PCR revealed the correct genotype. The results were also reproduced when a different reverse primer (5'-CGTGCACAAAGTTGTTCTGG-3'; GenBank accession no. HSU29185; position 25981–25962) was combined with the first hybridization probe system.
Our results suggest that a reannealing of equimolar amounts of the PCR product strands inhibited the binding of the detection probe to the valine genotype. However, the replacement of G with A in the methionine genotype may have allowed probe hybridization. The net result was a single melting peak for a heterozygous sample (Fig. 1C
, curve 1). The asymmetric PCR protocol with more forward than reverse primer allowed fast and specific genotyping of the human prion protein polymorphism at codon 129 with the hybridization probes we designed and obviated the need for redesign and expensive ordering of new probes. An assay for prion protein genotyping that apparently works under symmetric PCR conditions was published recently (5).
Since its introduction, many publications have shown the superior performance of the LightCyclerTM in rapid genotyping of single-nucleotide polymorphisms [e.g., see Refs. (6)(7)(8)]. Several reports have documented the robustness of this type of assay. However, these have not mentioned the optimization procedure for assay development and that changes in reaction conditions might cause misleading genotyping results. Our findings suggest the value of careful evaluation of new LightCycler genotyping assays and of controls for each genotype.
References
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