Detection of novel ALAD gene polymorphisms using denaturing high-performance liquid chromatography

Denaturing high-performance liquid chromatography (DHPLC), which is based o n the separation of mismatched DNA heteroduplexes, is one of the most promi sing techniques for detecting nucleotide polymorphisms. Lead is an importan t environmental toxicant that can impair the cardiovascular, central nervou s, renal, reproductive, and hematologic systems. Here we compare the sensit ivity and efficiency of DNA polymorphism detection in the delta -aminolevul inate dehydratase (ALAD) gene encoding the principal lead-binding protein i n humans by means of DHPLC and direct DNA sequencing of polymerase chain re action amplicons. In a sample of 48 unrelated Chinese women, five novel mut ations were discovered in intron 6 (G13298C), exon 7 (C13348T), intron 8 (C 13847T), intron 12 (C15096T), and the 3 ' untranslated region of exon 13 (A 15762C). The allele frequencies of C13298, T13348, T13847, T15096, and C157 62 alleles were 21.3%, 2.3%. 82.1%, 62.5%, and 1.1%, respectively. All five mutations were detected by both DHPLC and direct DNA sequencing. No previo usly reported missense ALAD mutations were found in this Chinese population . Our study confirms that DHPLC provides an accurate method for the rapid i dentification of single nucleotide polymorphisms.