Analysis of the BglI restriction fragment length polymorphism in the human factor VIII gene using "virtual PCR"--a novel approach employing the polymerase chain reaction in the absence of sequence information for the locus.
The BglI restriction fragment length polymorphism (RFLP) of the human factor VIII (FVIII) gene is potentially useful in linkage studies in haemophilia A. The sequence at the RFLP locus is not known, therefore it is not amenable to analysis by the polymerase chain reaction (PCR) and Southern blotting is required. We present a novel approach for analysis of the BglI RFLP using the PCR targeted to known sequence downstream in exon 26 of the FVIII gene. Briefly, the size of the genomic restriction fragment carrying the PCR target depends upon whether the RFLP site is present or absent. If fragments of the required size are isolated from a genomic digest and used as substrates in the exon 26 PCR, the generation of a product in one or other fraction indicates the upstream RFLP status. We have called this approach "virtual PCR", since PCR is used to obtain information about the RFLP without amplifying the locus itself.
Bowen
D J
DJ
Arthur Bloom Centre, Department of Haematology, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Hampton
K K
KK
D001483:Base Sequence; D002874:Chromosome Mapping; D017931:DNA Primers / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D005091:Exons; D005169:Factor VIII / Q000235:genetics*; D005260:Female; D008040:Genetic Linkage; D005819:Genetic Markers; D006467:Hemophilia A / Q000235:genetics; D006801:Humans; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length
D017931:DNA Primers; D005819:Genetic Markers; D005169:Factor VIII; D015252:Deoxyribonucleases, Type II Site-Specific; C018492:GCCNNNNNGGC-specific type II deoxyribonucleases