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 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.


CONCLUSIONS
This approach has applications for testing other medically relevant single-nucleotide polymorphisms.
Castley
Alison
A
Department of Clinical Immunology, Haematology, Royal Perth Hospital, Australia.
Higgins
Melinda
M
Ivey
John
J
Mamotte
Cyril
C
Sayer
David C
DC
Christiansen
Frank T
FT
D001769:Blood; D001800:Blood Specimen Collection; D005165:Factor V / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer; D005838:Genotype; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011516:Prothrombin / Q000235:genetics*
C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin