Fluorescent in situ hybridization of mitochondrial DNA and RNA

Abstract

To reveal nucleic acid localization in mitochondria, we designed molecular beacon fluorescent probes against: i) the light strand complementary to ND5 mitochondrial DNA (mtDNA) gene (annealing also to corresponding mRNA); ii) displacement (D) loop 7S DNA (annealing also to parallel heavy strand mtDNA and corresponding light strand transcript); iii) the proximal D-loop heavy strand displaced by the light strand promoter minor RNA. Confocal microscopy demonstrated ND5 probe spreading (less for other probes) in mitochondrial reticulum tubules but upon RNase A treatment all probes contoured mtDNA nucleoid localization.

DNase I spread the signal over mitochondrial tubules. Future applications are discussed.

Keywords

• mitochondrial DNA and RNA
• nucleoids of mitochondrial DNA
• molecular beacon fluorescent hybridization probes
• confocal microscopy
• transcription and replication intermediates