The sequence analysis of the 3′ end of intron 15 from human LDL receptor gene and its application in the study of gene diagnosis for familial hypercholesterolaemia

To understand intron 15 of human LDL receptor gene, the DNA fragments from exon 15 to exon 16 and the 3′ end of intron 15 were amplified with long chain PCR and anchored PCR The 3′ end of intron 15 was sequenced with Dynalbeads-Streptavidin Solid Phase technique. The sequence analysis showed that the 3′ end of intron 15 contained the 3′ splicing site and the branch site at 31 nucleotides upstream of the 3′ end. Besides the authentic branch site, it is possible thatthe 3′ end of intron 15 contains a cryptic site (GCCTCAC) at 20 nucleotides upstream of the 3′ end. The sequences suggest that the PvuII polymorphism at Intron 15 is caused by the T-C substitution. According to the sequences of the 3′ end, the new PCR-RFLP protocol for detection of PvuII polymorphism at intron 15 was developed. Using this protocol a representative familial hypercholesterolaemia family was identified with linkage analysis of PvuII polymorphism at intron 15.