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 h aemophilia A. The sequence at the RFLP locus is not known, therefore i t is not amenable to analysis by the polymerase chain reaction (PCR) a nd Southern blotting is required. We present a novel approach for anal ysis of the BglI RFLP using the PCR targeted to known sequence downstr eam in exon 26 of the FVIII gene. Briefly, the size of the genomic res triction fragment carrying the PCR target depends upon whether the RFL P site is present or absent. If fragments of the required size are iso lated 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 up stream RFLP status. We have called this approach ''virtual PCR'', sinc e PCR is used to obtain information about the RFLP without amplifying the locus itself.