Journal: Leukemia research
Article Title: Identification of Mitochondrial Genome Concatemers in AIDS-associated lymphomas and lymphoid cell lines
Figure Lengend Snippet: Isolation and characterization of EL cccDNA A) Episomal cccDNA was purified from EL cells by alkaline lysis and CsCl-EtBr gradient centrifugation. Density of gradient fractions decreases from left to right. Fractions were analyzed by agarose gel electrophoresis followed by SYBRGreen® staining. Figure is a representative of 7 independent experiments. *High molecular weight genomic DNA; **genomic broken linear DNA front. B) SYBRGreen™ staining of purified total cccDNA fractions from EL and normal T-cells. C) Southern Blotting of DNA (from B) using a probe of radiolabeled human mtDNA-specific oligonucleotides. D) Electron Microscopy of EL cccDNA. The contour length of the concatemeric mtDNA was measured using the Image Processing and Analysis In Java (ImageJ) software from the NIH. DNA contour length was calculated to be around 10.3 μm. Similar results were obtained measuring the contour length of 50 other molecules. E) Banding pattern of fully digested EL cccDNA with BamHI (10U) (mtDNA single cutter) at 30, 60 and 90 min resembles that of linear human mtDNA monomer (16.5 kbp). F) Partial digestion of EL cccDNA with BamHI (0.1U) stopped at increasing time points (5s, 10s, 20s, 40s, 1min, 2min, 5min, 10min, 20min, 40 min, 1hr). After 5 minutes, two bands of linear DNA are observed representing putative human mtDNA monomers and dimers (16.5 and 33 kbp respectively).
Article Snippet: The gel was stained with SYBRGreen® Nucleic Acid Stain (Lonza Rockland, Inc., Rockland, ME) for 1 hr, de-stained in 10mM Tris 1mM EDTA for 30 minutes and visualized using a Molecular Dynamics STORM Phosphor Imager (Model 860-PC, Amersham Biosciences, Piscataway, NJ).
Techniques: Isolation, Purification, Alkaline Lysis, Gradient Centrifugation, Agarose Gel Electrophoresis, Staining, Molecular Weight, Southern Blot, Electron Microscopy, Software