Comparative identity and homogeneity testing of the mtDNA HV1 region using denaturing gradient gel electrophoresis.
A denaturing gradient gel electrophoresis (DGGE) assay has been developed for comparative identity and homogeneity testing of the mtDNA HV1 region. A total of 49 pairs of sequences, each pair differing by a single unique polymorphism, were tested to verify the reliability of the assay. Discrimination between all pairings was achieved as judged by the resolution of the mismatch-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 heteroduplexes. Additional information derived from the technique includes the identification of co-amplifying contaminating or heteroplasmic samples in the independent samples lanes. Thirteen heteroplasmic samples, six at positions distinct from those analyzed in the pairwise comparison study, were analyzed and the heteroplasmic positions identified unambiguously by sequencing the excised bands. The technique constitutes a conceptually simple, accurate, and inexpensive test for determining whether two sequences match within the mtDNA HV1 region, while providing a more definitive control for the identification of co-amplifying contaminating or heteroplasmic sequences than is presently available.
Steighner
R J
RJ
Armed Forces DNA Identification Laboratory, Armed Forces Institute of Pathology, Rockville, MD, USA. SteighnerB@aol.com
Tully
L A
LA
Karjala
J D
JD
Coble
M D
MD
Holland
M M
MM
D000595:Amino Acid Sequence; D001483:Base Sequence; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005554:Forensic Medicine / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D004272:DNA, Mitochondrial