Clinical Chemistry
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Clinical Chemistry 45: 1925-1931, 1999;
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(Clinical Chemistry. 1999;45:1925-1931.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Activated Protein C Resistance: Automated Detection of the Factor V Leiden Mutation by Mismatch Hybridization

Karl R. Klinglera, Thomas Junold and Klaus Wielckens

a Author for correspondence. Fax 0221-478-5273.

Background: A single point mutation in the factor V gene has been demonstrated to be the cause of factor Va resistance to proteolytic cleavage by activated protein C. Knowledge of the patient's genetic disposition is of great importance in situations such as pregnancy, surgery, use of oral contraceptives, and immobilization.

Methods: We have developed a rapid, automated test for the detection of the factor V mutation that makes use of differences in thermal stability between perfect-match and non-perfect-match hybrids. A DNA fragment spanning the mutation is amplified with a biotin-labeled primer. Ruthenium-labeled oligonucleotides, perfectly matching either the biotinylated wild-type strand or the biotinylated mutation strand, are added. Heating to 95 °C and subsequent cooling lead to the formation of double-stranded DNA. Under the conditions chosen, ruthenium-labeled oligonucleotides form stable, double-stranded DNA with the biotinylated strand only if both strands perfectly match each other. The ruthenium signal is measured on a modified Elecsys 1010 system (Roche Diagnostics).

Results: The ratio between the signals obtained with perfectly matching and non-perfectly matching oligonucleotides reflects the genetic status. Analyzed samples can be divided into three nonoverlapping groups based on these ratios. We confirmed the reliability of the method by analyzing several samples of known genetic status; the results were identical in every single instance.

Conclusions: The test discriminates unambiguously between the heterozygous and the homozygous states. Because of its low costs and easy handling, the assay is suitable for use in routine laboratories of clinical chemistry.






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