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Technical Briefs |
Department of Clinical Chemistry, Georg-August-University, Robert Koch Strasse 40, 37075 Goettingen, Germany
a author for
correspondence: fax 49-551-39-2955, e-mail nahsen@gwdg.de
Analytical procedures for the identification of point mutations in genomic DNA are finding increasing application in the clinical laboratory. As the demand for these analyses grows, so does the need for rapid, reliable, and easy methods to detect known point mutations. Prothrombotic mutations can be found in almost 25% of unselected patients referred for thrombophilia workup (1). These include the factor V (G1691A), prothrombin (G20210A), and methylenetetrahydrofolate reductase (MTHFR; C677T) mutations (2)(3)(4). Recently, methods have been published for genotyping both the factor V (5) and the MTHFR (6) mutations by rapid cycle PCR using the LightCyclerTM (Roche Molecular Biochemicals). We describe here a procedure for genotyping the prothrombin mutation using the LightCycler. In addition, we have modified the PCR methods to perform all three PCR analyses in parallel, using the same program on the LightCycler. When this method is combined with a rapid DNA extraction, results can be obtained within 60 min after a whole blood sample is received.
The appearance of a specific PCR product is monitored by adjacent
hybridization probes (a labeled primer may also serve as a probe), that
are usually designed to bind on one amplicon strand. The 3' end of one
probe is labeled with fluorescein (FLU), whereas the 5' end of an
adjacent probe is labeled with LC-Red640 (Roche Molecular Biochemicals)
as the anchor probe. When both probes hybridize in close proximity,
fluorescence resonance energy transfer (FRET) occurs, producing a
specific fluorescence emission of LC-Red as a result of FLU excitation.
Increasing the temperature during fluorescence reading yields a
temperature/fluorescence curve from which the melting point of the
probe can be derived. When the appropriate conditions are chosen, the
mismatch under the detection probe caused by a single point mutation
leads to
References
The following articles in journals at HighWire Press have cited this article:
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  C. Sucker, C. Kurschat, F. Farokhzad, G. R. Hetzel, B. Grabensee, B. Maruhn-Debowski, R. Loncar, R. E. Scharf, and R. B. Zotz The TT Genotype of the C677T Polymorphism in the Methylentetrahydrofolate Reductase as a Risk Factor in Thrombotic Microangiopathies: Results From a Pilot Study Clinical and Applied Thrombosis/Hemostasis, June 1, 2009; 15(3): 283 - 288. [Abstract] [PDF]
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C. Sucker, C. Kurschat, G. R. Hetzel, B. Grabensee, B. Maruhn-Debowski, R. Loncar, L. Ostojic, R. E. Scharf, and R. B. Zotz The G1691A Mutation of the Factor V Gene (Factor V Leiden) and the G20210A Mutation of the Prothrombin Gene as Risk Factors in Thrombotic Microangiopathies Clinical and Applied Thrombosis/Hemostasis, June 1, 2009; 15(3): 360 - 363. [Abstract] [PDF]
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 E. Berge, K. B. F. Haug, E. Charlotte Sandset, K. Kristine Haugbro, M. Turkovic, and P. M. Sandset The Factor V Leiden, Prothrombin Gene 20210GA, Methylenetetrahydrofolate Reductase 677CT and Platelet Glycoprotein IIIa 1565TC Mutations in Patients With Acute Ischemic Stroke and Atrial Fibrillation Stroke, March 1, 2007; 38(3): 1069 - 1071. [Abstract] [Full Text] [PDF]
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 E. Rossou, A. Kouvatsi, C. Aslanidis, and C. Deltas Multiplex Molecular Diagnosis of MEFV Mutations in Patients with Familial Mediterranean Fever by LightCycler Real-Time PCR Clin. Chem., September 1, 2005; 51(9): 1725 - 1727. [Full Text] [PDF]
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M. S. Mahadevan and P. V. Benson Factor V Null Mutation Affecting the Roche LightCycler Factor V Leiden Assay Clin. Chem., August 1, 2005; 51(8): 1533 - 1535. [Full Text] [PDF]
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C. G. Tag, M.-C. Schifflers, M. Mohnen, A. M. Gressner, and R. Weiskirchen Atypical Melting Curve Resulting from Genetic Variation in the 3' Untranslated Region at Position 20218 in the Prothrombin Gene Analyzed with the LightCycler Factor II (Prothrombin) G20210A Assay Clin. Chem., August 1, 2005; 51(8): 1560 - 1561. [Full Text] [PDF]
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 K. Saxena, M. Ranalli, N. Khan, C. Blanchong, and S. B. Kahwash Fatal Stroke in a Child with Severe Iron Deficiency Anemia and Multiple Hereditary Risk Factors for Thrombosis Clinical Pediatrics, March 1, 2005; 44(2): 175 - 180. [PDF]
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S. Bortolin, M. Black, H. Modi, I. Boszko, D. Kobler, D. Fieldhouse, E. Lopes, J.-M. Lacroix, R. Grimwood, P. Wells, et al. Analytical Validation of the Tag-It High-Throughput Microsphere-Based Universal Array Genotyping Platform: Application to the Multiplex Detection of a Panel of Thrombophilia-Associated Single-Nucleotide Polymorphisms Clin. Chem., November 1, 2004; 50(11): 2028 - 2036. [Abstract] [Full Text] [PDF]
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 N. von Ahsen and M. Oellerich The intronic prothrombin 19911A>G polymorphism influences splicing efficiency and modulates effects of the 20210G>A polymorphism on mRNA amount and expression in a stable reporter gene assay system Blood, January 15, 2004; 103(2): 586 - 593. [Abstract] [Full Text] [PDF]
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M. Erali, B. Schmidt, E. Lyon, and C. Wittwer Evaluation of Electronic Microarrays for Genotyping Factor V, Factor II, and MTHFR Clin. Chem., May 1, 2003; 49(5): 732 - 739. [Abstract] [Full Text] [PDF]
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A. Ruiz, G. Antinolo, I. Marcos, and S. Borrego Novel Technique for Scanning of Codon 634 of the RET Protooncogene with Fluorescence Resonance Energy Transfer and Real-Time PCR in Patients with Medullary Thyroid Carcinoma Clin. Chem., November 1, 2001; 47(11): 1939 - 1944. [Abstract] [Full Text] [PDF]
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M. L. Smit, B. A.J. Giesendorf, J. A.M. Vet, F. J.M. Trijbels, and H. J. Blom Semiautomated DNA Mutation Analysis Using a Robotic Workstation and Molecular Beacons Clin. Chem., April 1, 2001; 47(4): 739 - 744. [Abstract] [Full Text] [PDF]
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G. Endler, P. A. Kyrle, S. Eichinger, M. Exner, and C. Mannhalter Multiplexed Mutagenically Separated PCR: Simultaneous Single-Tube Detection of the Factor V R506Q (G1691A), the Prothrombin G20210A, and the Methylenetetrahydrofolate Reductase A223V (C677T) Variants Clin. Chem., February 1, 2001; 47(2): 333 - 335. [Full Text] [PDF]
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R. D. Press Detection of Prevalent Genetic Alterations Predisposing to Hemochromatosis and Other Common Human Diseases Clin. Chem., October 1, 2000; 46(10): 1526 - 1528. [Full Text] [PDF]
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F. A.J.T.M. van den Bergh, A. M. van Oeveren-Dybicz, and M. A.M. Bon Rapid Single-Tube Genotyping of the Factor V Leiden and Prothrombin Mutations by Real-Time PCR Using Dual-Color Detection Clin. Chem., August 1, 2000; 46(8): 1191 - 1195. [Full Text] [PDF]
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 S. Bleich, D. Degner, J. Wiltfang, J. M. Maler, P. Niedmann, S. Cohrs, A. Mangholz, J. Porzig, R. Sprung, E. Ruther, et al. ELEVATED HOMOCYSTEINE LEVELS IN ALCOHOL WITHDRAWAL Alcohol Alcohol., July 1, 2000; 35(4): 351 - 354. [Abstract] [Full Text] [PDF]
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M. A.M. Bon, A. van Oeveren-Dybicz, and F. A.J.T.M van den Bergh Genotyping of HLA-B27 by Real-Time PCR without Hybridization Probes Clin. Chem., July 1, 2000; 46(7): 1000 - 1002. [Full Text] [PDF]
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M. Nauck, M. M. Hoffmann, H. Wieland, and W. Marz Evaluation of the Apo E Genotyping Kit on the LightCycler, Clin. Chem., May 1, 2000; 46(5): 722 - 724. [Full Text] [PDF]
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P. S. Bernard and C. T. Wittwer Homogeneous Amplification and Variant Detection by Fluorescent Hybridization Probes Clin. Chem., February 1, 2000; 46(2): 147 - 148. [Full Text] [PDF]
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N. von Ahsen, M. Oellerich, and E. Schutz Use of Two Reporter Dyes without Interference in a Single-Tube Rapid-Cycle PCR: {alpha}1-Antitrypsin Genotyping by Multiplex Real-Time Fluorescence PCR with the LightCycler Clin. Chem., February 1, 2000; 46(2): 156 - 161. [Abstract] [Full Text] [PDF]
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T. Aoshima, Y. Sekido, T. Miyazaki, M. Kajita, S. Mimura, K. Watanabe, K. Shimokata, and T. Niwa Rapid Detection of Deletion Mutations in Inherited Metabolic Diseases by Melting Curve Analysis with LightCycler Clin. Chem., January 1, 2000; 46(1): 119 - 122. [Full Text] [PDF]
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N. von Ahsen, M. Oellerich, V. W. Armstrong, and E. Schutz Application of a Thermodynamic Nearest-Neighbor Model to Estimate Nucleic Acid Stability and Optimize Probe Design: Prediction of Melting Points of Multiple Mutations of Apolipoprotein B-3500 and Factor V with a Hybridization Probe Genotyping Assay on the LightCycler Clin. Chem., December 1, 1999; 45(12): 2094 - 2101. [Abstract] [Full Text] [PDF]
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K. R. Klingler, T. Junold, and K. Wielckens Activated Protein C Resistance: Automated Detection of the Factor V Leiden Mutation by Mismatch Hybridization Clin. Chem., November 1, 1999; 45(11): 1925 - 1931. [Abstract] [Full Text] [PDF]
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