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This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: clinchem.2005.055327v1 51/11/2025    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Castley, A. Articles by Christiansen, F. T. Search for Related Content PubMed PubMed Citation Articles by Castley, A. Articles by Christiansen, F. T. Related Collections Molecular Diagnostics and Genetics

Clinical Applications of Whole-Blood PCR with Real-Time Instrumentation

Departments of¹ Clinical Immunology and Biochemical Genetics, and² Haematology, Royal Perth Hospital, Perth, Australia.
³ School of Surgery and Pathology, University of Western Australia, Perth, Australia.

^aAddress correspondence to this author at: Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Perth, Australia 6000. Fax 61-08-92242920; e-mail Frank.Christiansen{at}health.wa.gov.au.

Background: As the genetic basis of many human diseases is being discovered, there is increasing need for the detection of single-nucleotide polymorphisms/mutations in medical laboratories. We describe an innovative approach that combines PCR amplification directly on whole blood and real-time detection PCR technology (WB-RTD PCR).

Methods: We compared WB-RTD PCR with the method for extracted DNA-RTD PCR for the detection of mutations in the prothrombin (n = 94), factor V Leiden (n = 49), and hemochromatosis (n = 22) genes. Mutation detection on the Roche LightCycler was based on use of fluorescence resonance energy transfer (FRET) probes and melting curve analysis. We also compared the WB-RTD PCR on the LightCycler and the ABI Prism^TM 7700 sequence detection system with minor groove– binding nonfluorescent quencher probes.

Results: We obtained complete concordance between both methods in assigning genotypes. We also demonstrated that the WB-RTD PCR method can be performed on real-time PCR instruments from Applied Biosystems and the LightCycler. Omission of the need for DNA extraction and gel electrophoresis allowed substantial labor and cost savings with this method.

Conclusion: This approach has applications for testing other medically relevant single-nucleotide polymorphisms.

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