Comparative identity and homogeneity testing of the mtDNA HV1 region usingdenaturing gradient gel electrophoresis

A denaturing gradient gel electrophoresis (DGGE) assay has been developed f or comparative identity and homogeneity testing of the mtDNA HV1 region. A total of 49 pairs of sequences, each pair differing by a single unique poly morphism, were tested to verify the reliability of the assay. Discriminatio n between all pairings was achieved as judged by the resolution of the mism atch-containing heteroduplexes from the fully base-paired homoduplexes. In all but two pairings, resolution of the fully base-paired homoduplexes was also obtained. Sequence pairs differing by multiple polymorphisms were also tested and resulted in a greater separation between the homo- and heterodu plexes. Additional information derived from the technique includes the iden tification of co-amplifying contaminating or heteroplasmic samples in the i ndependent samples lanes. Thirteen heteroplasmic samples, six at positions distinct from those analyzed in the pairwise comparison study, were analyze d and the heteroplasmic positions identified unambiguously by sequencing th e excised bands. The technique constitutes a conceptually simple, accurate, and inexpensive test for determining whether two sequences match within th e mtDNA HV1 region, while providing a more definitive control for the ident ification of co-amplifying contaminating or heteroplasmic sequences than is presently available.