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Rapid detection of the factor V Leiden mutation by real-time PCR with TagMan minor groove binder probes.

To the Editor:

Individuals heterozygous for the factor V Leiden mutation have a sevenfold higher risk to develop venous thrombosis than wild-type (WT) individuals (1). The allele frequency of factor V Leiden in Europeans is ~4.4%,and the mutation is currently considered as the most important genetic risk factor for venous thrombosis (2). Various methods have been published to detect the factor V Leiden mutation by real-time PCR using hybridization probes (3-5) and TagMan probes (6, 7). Here we describe a robust and convenient procedure for the detection of the factor V Leiden mutation that uses TagMan probes conjugated to a minor groove binder (MGB) group.

TagMan probes are degraded during amplification by the 5'~3' exo nuclease activity of the polymerase. During degradation of the probe, the fluorescent group is separated from the quencher, leading to increased fluorescence. For allelic discrimination, probes matching the WT and the mutant allele are conjugated to a different fluorescent group. Discrimination between alleles is based on the difference in melting temperature ([DELTA}[T.sub.m]) between the differently labeled match and mismatch probes. For TagMan assays, relatively long probes are required that remain annealed during the extension phase of the PCR. A 1-bp mismatch in such a probe may lead to a small [DELTA][T.sub.m] and subsequent difficult allelic discrimination. DNA probes conjugated to a MGB group form hyperstabilized duplexes with complementary DNA by folding of the MGB group into the duplex, giving a higher [T.sub.m] (8, 9). The conjugation of a MGB group therefore allows the design of shorter probes with an increased specificity attributable to increased mismatch discrimination (9).

For detection of the factor V Leiden mutation, we designed MGB probes with a length of 17 bases, which is >25% shorter than the length (23-24 bases) of previously published TagMan probes for factor V Leiden detection (6, 7) without a MGB group. In addition, the probes were designed with the mutation site located in the MGB region to achieve the maximum ability to discriminate alleles (9).

The following primer and probe sequences were designed with Primer Express software (Applied Biosystems) and synthesized by Applied Biosystems: forward primer, 5'-GCC TCT GGG CTA ATA GGA CTA CTT C-3'; reverse primer, 5'TTT CTG AAA GGT TAC TTC AAG GAC AA-3'; WT factor V probe, FAM-ACC TGT ATT CCT CGC CTnfq-MGB, and factor V Leiden probe, VIC[R]-ACC TGT ATT CCT TGC CTnfq-MGB. The primers generate a 97-bp PCR product. The 5'-fluorescently labeled probes [6-carboxyfluorescein (FAM) or VIC] were conjugated to a nonfluorescent quencher (nfq) and an MGB group at the 3' end.

DNA was isolated from 200 [micro]L of EDTA-treated blood with the QIAamp DNA Mini Kit (Qiagen) according to the instructions of the manufacturer. The DNA was eluted in 200 [micro]L of elution buffer and stored at -20 [degrees]C. PCR was performed in a final reaction volume of 25 [micro]L, which contained 12.5 [micro]L of 2x TagMan Universal PCR Master Mix (Applied Biosystems), 450 n1V1 each primer, 200 n1V1 each probe, and 5 [micro]L of DNA solution. PCR was performed in MicroAmp optical 96-well plates with optical adhesive covers (Applied Biosystems). Amplification and detection were carried out with an ABI prism 7000 sequence detection system (Applied Biosystems). The amplification conditions were 2 min at 50 [degrees]C for AmpErase uracil-N-glycosylase activity and 10 min at 95 [degrees]C for AmpliTaq Gold activation, followed by 40 cycles of 15 s at 95 [degrees]C for denaturation and 1 min at 60 [degrees]C for annealing and extension. After amplification, endpoint detection of fluorescence was performed at 60 [degrees]C. The fluorescence data were analyzed with the allelic discrimination software of the ABI prism 7000 instrument.

The genotypes of 60 clinical samples were analyzed retrospectively. These samples were submitted to the laboratory for factor V Leiden mutation detection, and the genotype was initially established by PCR followed by restriction fragment analysis based on the method described by Koeleman et al. (10). Real-time factor V Leiden mutation detection was performed in duplicate. After amplification, four fluorescence patterns are expected. In samples from homozygous WT factor V patients, FAM fluorescence attributable to degradation of the WT probe will be present. In samples from patients homozygous for factor V Leiden, VIC fluorescence attributable to degradation of the factor V Leiden probe will be present. In samples from heterozygous patients, both FAM and VIC fluorescence will be detected. In PCRs to which no DNA was added, no fluorescence is expected except for the background signal. Fig. 1 shows that these four groups are clearly detected without overlap. The established genotypes were 100% identical to the results obtained with the method of Koeleman et al. (10).

[FIGURE 1 OMITTED]

The amount of isolated DNA added to the PCR will vary with the amount of leukocytes in the blood. To investigate the effect of various amounts of DNA on the performance of the assay, we made dilution series of DNA from all genotypes and added the DNA to the PCR in amounts ranging from 9.4 to 300 ng. A standard reaction is expected to contain ~150 ng of DNA. Although there was some variation in the amount of fluorescence, allelic discrimination was reliable with all amounts of input DNA (results not shown).

In conclusion, this real-time PCR method with TagMan MGB probes is a robust and convenient way to de termine the presence of the factor V Leiden mutation. The use of TagMan MGB probes gives clear discrimination of genotypes independent of the amount of input DNA. In addition to unambiguous discrimination, the real-time assay with MGB probes has the advantages of other real-time assays, such as decreased assay time and contamination risks. Furthermore, the absence of ambiguous results eliminates the need for repetitive analysis of a sample. Designing primers and probes with Primer Express software (Applied Biosystems) makes it possible to run PCRs under universal conditions, meaning that other single-nucleotide polymorphisms can be detected in the same run. The combination of TagMan MGB probes with universal assay conditions allows an efficient workflow when the number of different single-nucleotide polymorphisms that are detected in the clinical laboratory increases.

We acknowledge Henk Ruven (St. Antonius Hospital, Nieuwegein, The Netherlands) for providing some reference samples. We also thank Steven Thijsen (Diakonessenhuis, Utrecht, The Netherlands) for critical reading of the manuscript.

References

1. Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in patients homorygous for factor V Leiden (activated protein C resistance). Blood 1995;85:1504-8.

2. Rees DC, Cox M, Clegg JB. World distribution of factor V Leiden. Lancet 1995;346:1133-4.

3. Lay MJ, Wittwer CT. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin Chem 1997;43:2262-7.

4. von Ahsen N, Oellerich M, Schutz E. A method for homogeneous color-compensated genotyping of factor V (G1691A) and methylenetetrahydrofolate reductase (C677T) mutations using real-time multiplex fluorescence PCR. Clin Biochem 2000;33:535-9.

5. van den Bergh FA, Oeveren-Dybicz AM, Bon MA. Rapid single-tube genotyping of the factor V Leiden and prothrombin mutations by real-time PCR using dual-color detection. Clin Chem 2000;46:1191-5.

6. Happich D, Schwaab R, Hanfland P, Hoernschemeyer D. Allelic discrimination of factor V Leiden using a 5' nuclease assay. Thromb Haemost 1999;82:1294-6.

7. Sanders SJ. Factor V. Leiden genotyping using real-time fluorescent polymerase chain reaction. Mol Cell Probes 2000;14:249-53.

8. Kumar S, Reed MW, Gamper HB Jr, Gorn W, Lukhtanov EA, Foti M, et al. Solution structure of a highly stable DNA duplex conjugated to a minor groove binder. Nucleic Acids Res 1998; 26:831-8.

9. Kutyavin IV, Afonina IA, Mills A, Gorn W, Lukhtanov EA, Belousov ES, et al. 3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 2000;28:655-61.

10. Koeleman BP, Reitsma PH, Allaart CF, Bertina RM. Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficient families. Blood 1994;84:1031-5.

Rianne Ludererl *

Alice Verheul (2)

Wouter Kortlandt (2)

(1) Unit Molecular Diagnostics

and (2) Department of Clinical Chemistry

Diakonessenhuis Utrecht

Bosboomstraat 1

3582 KE Utrecht, The Netherlands

* Author for correspondence. E-mail RLuderer@dialchuis.nl.
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Title Annotation:Letters
Author:Luderer, Rianne; Verheul, Alice; Kortlandt, Wouter
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Apr 1, 2004
Words:1365
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