Specific amplification of deleted mitochondrial DNA from a myopathic patient and analysis of deleted region with S1 nuclease.
Biochim Biophys Acta
; 1009(2): 151-5, 1989 Nov 02.
Article
em En
| MEDLINE
| ID: mdl-2804078
ABSTRACT
Heteroplasmy of the normal-sized and the deleted mitochondrial genome has been observed in mitochondrial myopathy. The deleted region of the genome in the skeletal muscle of a patient was analyzed both by the conventional Southern blot method and by the novel method of employing the combination of polymerase chain reaction and S1 nuclease digestion. The results obtained by these methods were compared. Southern hybridization using various mitochondrial DNA fragments localized the deletion from at least position 9020 to 14,955, but regions of uncertainty of 1 kb remained on both ends of the deletion. Using the polymerase chain reaction, a fragment from the deleted genome was specifically amplified by choosing a pair of primers surrounding the deletion, and two fragments adjacent to the starting and end of the deletion were amplified from the normal-sized genome. S1 nuclease analysis of the heteroduplexes formed among these fragments demonstrated that the deletion extended from positions 8650 +/- 50 to 15,660 +/- 60. This method does not require radioisotopes and, moreover, can determine the deleted region within 5 h, in contrast to the 2 days required by the conventional Southern blot analysis. These results indicate that the novel method is faster and more accurate than the conventional method for the determination of the deleted region of genome.
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Base de dados:
MEDLINE
Assunto principal:
DNA Mitocondrial
/
Oftalmoplegia
/
Amplificação de Genes
/
Reação em Cadeia da Polimerase
/
Deleção Cromossômica
/
Doenças Musculares
Limite:
Adult
/
Humans
/
Male
Idioma:
En
Revista:
Biochim Biophys Acta
Ano de publicação:
1989
Tipo de documento:
Article
País de afiliação:
Japão