Identification of a silent point mutation in the LDL-receptor gene by direct DNA sequencing.

To study the allelic variation at the human LDL-receptor gene locus in a number of individuals with familial hypercholesterolemia, a protocol was applied for direct sequence analysis of genomic DNA. The asymmetric polymerase chain reaction (PCR) was used to synthesize single-stranded DNA. Sequencing was carried out with modified T7 DNA polymerase (Sequenase version 2.0, United States Biochemical) after purification of the amplification product with a glass powder adhesion method. The method is sensitive enough to identify the heterozygous state of allelic variation and bypasses cumbersome cloning and subcloning procedures. Here we report the occurrence of a guaninine-to-adenine change in the codon for amino acid 655. However, the glycine residue is not replaced by the base change at this position, and the mutation observed here does not represent the underlying genetic defect of the LDL-receptor defect in this individual. Preliminary data suggest that this mutation represents a rare genetic variation.