Mapping of an origin of DNA replication near the transcriptional promoter of the human HPRT gene

A quantitative PCR method was used to map a functional origin of DNA replication in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in normal human fibroblasts. This PCR method measures the abundance of specific sequences in short fragments of newly replicated DNA from logarithmically growing cells. Quantitative measurements rely on synthetic molecules (competitors) that amplify with the same primer sets as the target molecules, but generate products of different sizes. This method was first utilized to determine the position of the replication origin near the lamin B2 gene (Giacca et al. [1994] Proc. Natl. Acad. Sci. U S A. 91:7119-7123). In the present study, primer sets were tested along a 1 6-kb region near exon 1 of the HPRT gene. The most abundant fragment was found to be located in the first intron of HPRT, just downstream of the promoter and exon 1 of the gene, and approximately 3.5 kb upstream of a previously reported autonomously replicating sequence (Sykes et al. [1988] Mol. Gen. Genet. 212:301-309). J. Cell. Biochem. 85: 346-356, 2002. (C) 2002 Wiley-Liss, Inc.