Clinical applications of whole-blood PCR with real-time instrumentation

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. (c) 2005 American Association for Clinical Chemistry.