Quantitation and detection of deletion in tumor mitochondrial DNA by microarray technique

Objective To develop a method to rapidly quantitate and detect dele tion 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 s amples of gastric cancerous tissues, which were simultaneously labeled with fluo rescin. The amplified products were verified by polyacrylamide gel electrophor esis (PAGE) and silver staining. Then,17 pairs of overlapping primers of mtDNA were designed and their PCR products were used as mitochondrial probes. They wer e spotted onto amino-slides as microarray and hybridized. Hybridization image w as scanned with GeneTAC TM laser, mtDNA copy number was counted by ScanAnal yzer software. Results PAGE analysis showed that the designed probes were quite reason able and strongly specific. The modified PCR method was efficient to amplify the whole mitochondrial genome with high-yield specific bands. The hy bridiz ing spots were distinct, and background was clear. The signals of negative probe s were close to those of background, and there was no significant difference bet ween them ( P 0.05). The results were identical to those in the designed expe rime nt. There were no significant differences between the results when the same samp le of blood leucocytes or cancer tissues repeatedly examined with the same posit ive probes ( P 0.05), while there were significant differences when different typ es of samples were examined ( P 0.01). The hybridizing signals were stable an d most of the data distributed in the range of mean±2xSD. Conclusion The method her e reported can rapidly, correctly and massively determine whether there exist sp ecial deletion and/or quantitative changes of mtDNA in patients with tumors. It will be helpful for the study of the relationship between mtDNA alter ation and tumor development.