OBJECTIVE: To develop a method to rapidly quantitate and detect deletion of mitochondrial DNA (mtDNA) by microarray technique as a tool to study its relationship to tumorigenesis. METHODS: A modified PCR was used to amplify full length mtDNA sequence in two samples of normal human blood leukocytes and five samples of gastric cancerous tissues, which were simultaneously labeled with fluorescin. The amplified products were verified by polyacrylamide gel electrophoresis (PAGE) and silver staining. Then, 17 pairs of overlapping primers of mtDNA were designed and their PCR products were used as mitochondrial probes. They were spotted onto amino-slides as microarray and hybridized. Hybridization image was scanned with GeneTAC laser, mtDNA copy number was counted by ScanAnalyzer software. RESULTS: PAGE analysis showed that the designed probes were quite reasonable and strongly specific. The modified PCR method was efficient to amplify the whole mitochondrial genome with high-yield specific bands. The hybridizing spots were distinct, and background was clear. The signals of negative probes were close to those of background, and there was no significant difference between them (P > 0.05). The results were identical to those in the designed experiment. There were no significant differences between the results when the same sample of blood leucocytes or cancer tissues repeatedly examined with the same positive probes (P > 0.05), while there were significant differences when different types of samples were examined (P < 0.01). The hybridizing signals were stable and most of the data distributed in the range of mean +/- 2xSD. CONCLUSION: The method here reported can rapidly, correctly and massively determine whether there exist special deletion and/or quantitative changes of mtDNA in patients with tumors. It will be helpful for the study of the relationship between mtDNA alteration and tumor development.
OBJECTIVE: To develop a method to rapidly quantitate and detect deletion of mitochondrial DNA (mtDNA) by microarray technique as a tool to study its relationship to tumorigenesis. METHODS: A modified PCR was used to amplify full length mtDNA sequence in two samples of normal human blood leukocytes and five samples of gastric cancerous tissues, which were simultaneously labeled with fluorescin. The amplified products were verified by polyacrylamide gel electrophoresis (PAGE) and silver staining. Then, 17 pairs of overlapping primers of mtDNA were designed and their PCR products were used as mitochondrial probes. They were spotted onto amino-slides as microarray and hybridized. Hybridization image was scanned with GeneTAC laser, mtDNA copy number was counted by ScanAnalyzer software. RESULTS: PAGE analysis showed that the designed probes were quite reasonable and strongly specific. The modified PCR method was efficient to amplify the whole mitochondrial genome with high-yield specific bands. The hybridizing spots were distinct, and background was clear. The signals of negative probes were close to those of background, and there was no significant difference between them (P > 0.05). The results were identical to those in the designed experiment. There were no significant differences between the results when the same sample of blood leucocytes or cancer tissues repeatedly examined with the same positive probes (P > 0.05), while there were significant differences when different types of samples were examined (P < 0.01). The hybridizing signals were stable and most of the data distributed in the range of mean +/- 2xSD. CONCLUSION: The method here reported can rapidly, correctly and massively determine whether there exist special deletion and/or quantitative changes of mtDNA in patients with tumors. It will be helpful for the study of the relationship between mtDNA alteration and tumor development.