Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson's syndrome lymphoblasts using a fluorogenic 5'-nuclease (TaqMan) real-time polymerase chain reaction assay and plasmid external calibration standards.
This study describes a multiplex real-time polymerase chain reaction (PCR) assay that quantifies total mitochondrial DNA (mtDNA(total)) and mtDNA bearing the 4977-base pair 'common deletion' (deltamtDNA4977) in lymphoblasts derived from an individual diagnosed with Pearson's syndrome. The method is unique in its use of plasmids as external quantification standards and its use of multiplex conditions. Standards are validated by comparison with purified mtDNA amplification curves and by the fact that curves are largely unaffected by nuclear DNA (nucDNA). Finally, slopes of standard curves and unknowns are shown to be similar to each other and to theoretical predictions. From these data, mtDNA(total) in these cells is calculated to be 3258 (+723/-592) copies per cell while deltamtDNA4977 averages 232 (+136/-86) copies per cell or 7% (+4.65/-2.81).
Pogozelski
Wendy Knapp
WK
Department of Chemistry, State University of New York College at Geneseo, Geneseo, NY 14454, USA. pogozels@geneseo.edu
Hamel
Christine J C
CJ
Woeller
Collynn F
CF
Jackson
William E
WE
Zullo
Steven J
SJ
Fischel-Ghodsian
Nathan
N
Blakely
William F
WF