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Clin Chem. 2005 Nov;51(11):2025-30. Epub 2005 Sep 15.

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

Author information

  • 1Department of Clinical Immunology, Haematology, Royal Perth Hospital, Australia.

Abstract

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 Prismtrade mark 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.

PMID:
16166168
[PubMed - indexed for MEDLINE]
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