Applications of constant denaturant capillary electrophoresis / high-fidelity polymerase chain reaction to human genetic analysis

Abstract

Constant denaturant capillary electrophoresis (CDCE) permits high-resolution separation of single-base variations occurring in an ˜100 bp isomelting DNA sequence based on their differential melting temperatures. By coupling CDCE for highly efficient enrichment of mutants with high-fidelity polymerase chain reaction (hifi PCR), we have developed an analytical approach to detecting point mutations at frequencies equal to or greater than 10—6 in human genomic DNA. In this article, we present several applications of this approach in human genetic studies. We have measured the point mutational spectra of a 100 bp mitochondrial DNA sequence in human tissues and cultured cells. The observations have led to the conclusion that the primary causes of mutation in human mitochondrial DNA are spontaneous in origin. In the course of studying the mitochondrial somatic mutations, we have also identified several nuclear pseudogenes homologous to the analyzed mitochondrial DNA fragment. Recently, through developments of the means to isolate the desired target sequences from bulk genomic DNA and to increase the loading capacity of CDCE, we have extended the CDCE/hifi PCR approach to study a chemically induced mutational spectrum in a single-copy nuclear sequence. Future applications of the CDCE/hifi PCR approach to human genetic analysis include studies of somatic mitochondrial mutations with respect to aging, measurement of mutational spectra of nuclear genes in healthy human tissues and population screening for disease-associated single nucleotide polymorphisms (SNPs) in large pooled samples.