MUTATIONAL SCANNING OF MITOCHONDRIAL-DNA BY 2-DIMENSIONAL ELECTROPHORESIS

An expedient, accurate, and cost-efficient test was developed to scan critical regions of the mitochondrial genome for all possible mutation s by two-dimensional DNA electrophoresis. The test involves a two-step multiplex PCR amplification: a long-distance PCR to amplify almost th e entire mitochondrial genome, which then serves as template for the a mplification of 25 short PCR fragments in two multiplex groups corresp onding to regions implicated in human diseases. The mixture of fragmen ts was subsequently subjected to two-dimensional electrophoretic separ ation, first by size in a nondenaturant polyacrylamide gel and then on the basis of basepair sequence in a denaturing gradient polyacrylamid e gel. This latter process of denaturing gradient gel electrophoresis is a most accurate form of mutation detection on the basis of differen ces in melting behavior of mutant and wildtype fragments. Evaluation o f the method using samples with known homoplasmic and heteroplasmic mu tations, as well as CEPH pedigrees to study segregation of polymorphic variants, indicated a very high accuracy; none of the previously iden tified mutations and polymorphisms escaped detection, and no erroneous segregation patterns of polymorphic variants were observed. In additi on, two variants were found to be novel mutations when analyzed by seq uence analysis. One of these novel mutations was a heteroplasmic mutat ion in the COXIII gene that was found to segregate to homoplasmy in th e next generation. Heteroplasmic mutations as low as 1% of mtDNA could still be detected. (C) 1998 Academic Press.