[A sensitive fluorescent assay for the detection and quantification of mitochondrial 3243 mutation].
A mutation at nucleotide 3243 in the mitochondrial DNA (mtDNA) specifying tRNALeu(UUR) has been shown to be related with diabetes mellitus. Since mtDNA shows heteroplasmy and its composition would change among various tissues, it is important to quantitate the percentage of mutant mtDNA precisely. Here we report a rapid and sensitive method to determine the mutant mtDNA. A fragment of mtDNA containing nucleotide 3243 was amplified by PCR using a rhodamine-labeled primer and the product was digested with ApaI. The PCR product from normal gene was resistant to ApaI digestion, while a mutation from A to G generated an ApaI site in the mutant mtDNA and its PCR product was cleaved into two fragments. The digested product was separated by acrylamide gel electrophoresis and each product was quantitated by a fluorescence analyzer. PCR analysis using standard mixtures of normal and mutant mtDNAs indicated that this method can be used to detect as little as 1% mutant mtDNA.
Yu
G S
GS
Endocrinology Section, R & D Center, BML, Inc., Kawagoe.
Otabe
S
S
Niwa
M
M
D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D003920:Diabetes Mellitus / Q000209:etiology; D005453:Fluorescence; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012235:Rhodamines
D017931:DNA Primers; D004272:DNA, Mitochondrial; D012235:Rhodamines