Abstract
Purpose: To quantify the degree of heteroplasmy of a mitochondrial DNA (mtDNA) mutation in Leber's hereditary optic neuropathy (LHON) a biplex Invader® assay was applied. Methods: To determine the optimum condition for the Invader® assay, mtDNAs were assayed in various amounts of total DNA in 1-4-h incubations at 63°C. To evaluate the suitability of the Invader® assay to detect the three mutations, G3460A, G11778A, and T14484C, 10 ng of DNAs from 224 patients with bilateral optic atrophy was assayed. To quantify mtDNA heteroplasmy, a standard curve of known mixture ratios of mutation against calculation by the Invader® assay was constructed. Seventy-two of the 224 patients had one of the three mutations, which corresponded with the mutation detected earlier by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. The percentages of mutant mtDNAs were calculated by the Invader® assay in five heteroplasmic families, including 30 individuals with the G11778A mutation. The results were compared with those calculated earlier by labeled polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis. Results: In 1-8 ng of DNA, the fluorescence intensity increased near linearly during a 4-h assay. With more than 16 ng of DNA, the intensities were saturated even at the 2-h assay. A linear relationship was observed between the results obtained from separate mixtures and from the Invader® assay analysis. Because two fluorescent intensities are not always the same, one of the two intensities was modified to adjust to that of the other. Complete concordance was observed between PCR-RFLP analysis and Invader® assay genotyping for the 224 patients. Results of percentage of heteroplasmy in five LHON families obtained by the Invader® assay were consistent with those by the PCR-SSCP analysis. Conclusions: Invader® assay is a simple, rapid, and reliable method of genotyping mtDNA mutations as well as quantifying heteroplasmy simultaneously under optimum conditions.
| Original language | English |
|---|---|
| Pages (from-to) | 268-276 |
| Number of pages | 9 |
| Journal | Clinical Biochemistry |
| Volume | 37 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2004 Apr |
Fingerprint
Keywords
- G11778A mutation
- G3460A mutation
- Heteroplasmy
- Invader technology
- Leber's hereditary optic neuropathy
- Mitochondrial DNA
- T14484CA mutation
ASJC Scopus subject areas
- Clinical Biochemistry
Cite this
Rapid quantification of the heteroplasmy of mutant mitochondrial DNAs in Leber's hereditary optic neuropathy using the Invader technology. / Mashima, Yukihiko; Nagano, Makoto; Funayama, Tomoyo; Zhang, Qiang; Egashira, Tohru; Kudo, Jun; Shimizu, Nobuyoshi; Oguchi, Yoshihisa.
In: Clinical Biochemistry, Vol. 37, No. 4, 04.2004, p. 268-276.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Rapid quantification of the heteroplasmy of mutant mitochondrial DNAs in Leber's hereditary optic neuropathy using the Invader technology
AU - Mashima, Yukihiko
AU - Nagano, Makoto
AU - Funayama, Tomoyo
AU - Zhang, Qiang
AU - Egashira, Tohru
AU - Kudo, Jun
AU - Shimizu, Nobuyoshi
AU - Oguchi, Yoshihisa
PY - 2004/4
Y1 - 2004/4
N2 - Purpose: To quantify the degree of heteroplasmy of a mitochondrial DNA (mtDNA) mutation in Leber's hereditary optic neuropathy (LHON) a biplex Invader® assay was applied. Methods: To determine the optimum condition for the Invader® assay, mtDNAs were assayed in various amounts of total DNA in 1-4-h incubations at 63°C. To evaluate the suitability of the Invader® assay to detect the three mutations, G3460A, G11778A, and T14484C, 10 ng of DNAs from 224 patients with bilateral optic atrophy was assayed. To quantify mtDNA heteroplasmy, a standard curve of known mixture ratios of mutation against calculation by the Invader® assay was constructed. Seventy-two of the 224 patients had one of the three mutations, which corresponded with the mutation detected earlier by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. The percentages of mutant mtDNAs were calculated by the Invader® assay in five heteroplasmic families, including 30 individuals with the G11778A mutation. The results were compared with those calculated earlier by labeled polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis. Results: In 1-8 ng of DNA, the fluorescence intensity increased near linearly during a 4-h assay. With more than 16 ng of DNA, the intensities were saturated even at the 2-h assay. A linear relationship was observed between the results obtained from separate mixtures and from the Invader® assay analysis. Because two fluorescent intensities are not always the same, one of the two intensities was modified to adjust to that of the other. Complete concordance was observed between PCR-RFLP analysis and Invader® assay genotyping for the 224 patients. Results of percentage of heteroplasmy in five LHON families obtained by the Invader® assay were consistent with those by the PCR-SSCP analysis. Conclusions: Invader® assay is a simple, rapid, and reliable method of genotyping mtDNA mutations as well as quantifying heteroplasmy simultaneously under optimum conditions.
AB - Purpose: To quantify the degree of heteroplasmy of a mitochondrial DNA (mtDNA) mutation in Leber's hereditary optic neuropathy (LHON) a biplex Invader® assay was applied. Methods: To determine the optimum condition for the Invader® assay, mtDNAs were assayed in various amounts of total DNA in 1-4-h incubations at 63°C. To evaluate the suitability of the Invader® assay to detect the three mutations, G3460A, G11778A, and T14484C, 10 ng of DNAs from 224 patients with bilateral optic atrophy was assayed. To quantify mtDNA heteroplasmy, a standard curve of known mixture ratios of mutation against calculation by the Invader® assay was constructed. Seventy-two of the 224 patients had one of the three mutations, which corresponded with the mutation detected earlier by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. The percentages of mutant mtDNAs were calculated by the Invader® assay in five heteroplasmic families, including 30 individuals with the G11778A mutation. The results were compared with those calculated earlier by labeled polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis. Results: In 1-8 ng of DNA, the fluorescence intensity increased near linearly during a 4-h assay. With more than 16 ng of DNA, the intensities were saturated even at the 2-h assay. A linear relationship was observed between the results obtained from separate mixtures and from the Invader® assay analysis. Because two fluorescent intensities are not always the same, one of the two intensities was modified to adjust to that of the other. Complete concordance was observed between PCR-RFLP analysis and Invader® assay genotyping for the 224 patients. Results of percentage of heteroplasmy in five LHON families obtained by the Invader® assay were consistent with those by the PCR-SSCP analysis. Conclusions: Invader® assay is a simple, rapid, and reliable method of genotyping mtDNA mutations as well as quantifying heteroplasmy simultaneously under optimum conditions.
KW - G11778A mutation
KW - G3460A mutation
KW - Heteroplasmy
KW - Invader technology
KW - Leber's hereditary optic neuropathy
KW - Mitochondrial DNA
KW - T14484CA mutation
UR - http://www.scopus.com/inward/record.url?scp=1542346402&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=1542346402&partnerID=8YFLogxK
U2 - 10.1016/j.clinbiochem.2003.11.011
DO - 10.1016/j.clinbiochem.2003.11.011
M3 - Article
C2 - 15003728
AN - SCOPUS:1542346402
VL - 37
SP - 268
EP - 276
JO - Clinical Biochemistry
JF - Clinical Biochemistry
SN - 0009-9120
IS - 4
ER -