FAST AMPLIFICATION OF THE LOW-DENSITY-LIPOPROTEIN RECEPTOR GENE AND DETECTION OF A LARGE DELETION BY MEANS OF LONG POLYMERASE CHAIN-REACTION

To demonstrate the usefulness of Long PCR in analyzing gene structures and large deletions we have developed a method to amplify the entire LDL receptor gene, including the promoter region and intron 1. This op ens new ways for studies of the gene and allows the detection of certa in LDL receptor-specific deletions. For the amplification of the LDL r eceptor gene, spanning approximately 45.5 x 10(3) bases and divided in to 18 exons and 17 introns, we have designed overlapping PCR products (ranging from 4 to 16 x 10(3) bases in length), which can be amplified simultaneously overnight for fast results. It was possible to positiv ely identify two samples from heterozygote carriers of the ''5 kB Fren ch-Canadian'' deletion using this method. As a side result the length of intron 1 of the LDL receptor gene could be established to be approx imately 9.5 x 10(3) bases. The method is sensitive enough to detect de letions in 1 : 10 mixes of positive control with wildtype DNA.