title: Restriction fragment analysis as a source of error in detection of heteroplasmic mtDNA mutations.
abstract: The transition from A to G at nt 5656 (5656A-->G) in mitochondrial DNA has been suggested to be a pathogenic mutation and, furthermore, a heteroplasmic one. We found that the mutation was present in 14 out of 83 healthy controls from northern Finland and that 5656A-->G was exclusively associated with mtDNA haplogroup U. Interestingly, 5656A-->G appeared to be heteroplasmic in NheI digestion of PCR fragments that were amplified by using a mismatched oligonucleotide primer creating a digestion site in the presence of the mutant variant. However, we did not detect the wild type genome in clones from such a sample and subsequent experiments revealed that the apparent heteroplasmy was due to inhibition of NheI by NaCl. Our results suggest that 5656A-->G is a polymorphism and it may be highly characteristic for Finns. Furthermore, new heteroplasmic mutations identified by restriction fragment analysis should be adequately controlled for any false positive results that may be due to incomplete digestion.
authors.affiliation: Department of Neurology, University of Oulu, P.O. Box 5000, 90401, Oulu, Finland.
authors.forename: S
authors.initials: S
authors.lastname: Finnilä
authors.forename: I E
authors.initials: IE
authors.lastname: Hassinen
authors.forename: K
authors.initials: K
authors.lastname: Majamaa
mesh_terms: D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000187:drug effects / Q000378:metabolism; D005189:False Positive Reactions; D006239:Haplotypes; D006801:Humans; D009154:Mutation; D017354:Point Mutation; D011110:Polymorphism, Genetic; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: D004272:DNA, Mitochondrial; D012965:Sodium Chloride; D004247:DNA; D004262:DNA Restriction Enzymes; C067043:endodeoxyribonuclease NheI; D015252:Deoxyribonucleases, Type II Site-Specific