Factor V Leiden detection by polymerase chain reaction-restriction fragment length polymorphism with mutagenic primers in a multiplex reaction with Pro G20210A--a novel technique.
Factor V Leiden (FVL) R506Q and Prothrombin G20210A are clinically important genetic mutations associated with increased susceptibility to venous thrombosis. The objective of our study was to design a polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) reaction that allows simultaneous detection of these two mutations. The reaction can be used in routine diagnostic settings. We have analysed 4504 alleles for each mutation with a mutagenic primer-based PCR system with a low failure rate. The system eliminates the false positive FVL G1691A results associated with other PCR/RFLP caused by rare confounding mutations adjacent to restriction endonuclease recognition sites. This multiplex PCR/RFLP reaction is rapid, robust and dependable.
Department of Molecular Genetics, Arrowe Park Hospital, Arrowe Park Road, Upton, Wirral, Merseyside, CH49 5PE, UK. john.welbourn@whnt.nhs.uk
J T
JT
Welbourn
S
S
Maiti
J
J
Paley
D020413:3' Untranslated Regions / Q000235:genetics*; D020016:Activated Protein C Resistance / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D000483:Alleles; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005165:Factor V / Q000032:analysis / Q000235:genetics*; D005260:Female; D006801:Humans; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D011247:Pregnancy; D011250:Pregnancy Complications, Hematologic / Q000097:blood / Q000175:diagnosis*; D011516:Prothrombin / Q000235:genetics*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D013379:Substrate Specificity; D019851:Thrombophilia / Q000097:blood / Q000175:diagnosis* / Q000235:genetics
D020413:3' Untranslated Regions; D017931:DNA Primers; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes