Use of a PCR-based amplification analysis as a substitute for the Southern blot method to determine the C4A and C4B...

The human C4 complement components of the C4 gene are encoded by two genes, C4A and C4B, located on chromosome 6p21.3 of the major histocompatibility complex (MHC) of the human leukocyte antigen (HLA) class III region. Genetic determination of these two genes was by the Southern blot method: the 276- and 191-bp NlaIV fragments represent the C4A gene with the sequence, PCPVLP, at residues 1101-1106; the 467-bp NlaIV fragment represents the C4B gene with the sequence, LSPVIH, at residues 1101-1106. Here, we describe a PCR-based approach for differential amplification of the C4 genes adjacent to the respective CYP21A1P and CYP21A2, followed by NlaIV restriction digestion in a secondary PCR product and direct analysis by electrophoresis on an agarose gel to determine the C4A and C4B genes. From the results of this study, we concluded that 87% and 85% of the C4 genes adjacent to the CYP21A1P and CYP21A2 genes carried the C4A and C4B genes, respectively. The frequencies of the C4A and C4B genes comprising the C4 locus were 51.5 and 49%, respectively in this ethnic Chinese (Taiwanese) cohort. Since no radiolabelling application is involved, the protocol is reliable as a substitute for the Southern blot method for C4A and C4B determination.