Evaluation of the Roche LightCycler: a simple and rapid method for direct detection of factor V Leiden and prothrombin G20210A genotypes from blood samples without the need for DNA extraction.
The Roche LightCycler is a micro-volume thermocycler that combines extremely rapid polymerase chain reaction with fluorescence resonance energy transfer analysis of amplified products. We have evaluated the use of minimally processed blood samples for detection of two point mutations known to increase the risk of venous thromboembolism. Results from the LightCycler using the supernatant of diluted heated blood were compared with those gained by traditional methods based on polymerase chain reaction and restriction enzyme digestion. For factor V Leiden mutation, there was complete agreement between both methods in detection of wild-type (n = 82), heterozygous (n = 100) and homozygous (n = 18) genotypes. Similarly, the prothrombin G20210A mutation showed complete agreement for wild-type (n = 135), heterozygous (n = 63) and homozygous (n = 2) subjects.
Cooper
Peter C
PC
Sheffield Haemophilia and Thrombosis Centre, Royal Hallamshire Hospital, UK. peter.cooper@sth.nhs.uk
Cooper
Susan M
SM
Smith
Julie M
JM
Kitchen
Steven
S
Makris
Michael
M
D020413:3' Untranslated Regions / Q000235:genetics*; D020016:Activated Protein C Resistance / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D000483:Alleles; D004252:DNA Mutational Analysis / Q000295:instrumentation*; D005165:Factor V / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D005838:Genotype; D006801:Humans; D020125:Mutation, Missense; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D011516:Prothrombin / Q000235:genetics*; D019851:Thrombophilia / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D013997:Time Factors
D020413:3' Untranslated Regions; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin