title: A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA.
abstract: Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late 'point of no return' step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR's advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR's utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science.
authors.affiliation: Centre for Virology, Burnet Institute, 85 Commercial Road, Melbourne, Victoria 3004, Australia. dahoo@burnet.edu.au
authors.forename: David J
authors.initials: DJ
authors.lastname: Hooker
authors.forename: Masqura
authors.initials: M
authors.lastname: Mobarok
authors.forename: Jenny L
authors.initials: JL
authors.lastname: Anderson
authors.forename: Reena
authors.initials: R
authors.lastname: Rajasuriar
authors.forename: Lachlan R
authors.initials: LR
authors.lastname: Gray
authors.forename: Anne M
authors.initials: AM
authors.lastname: Ellett
authors.forename: Sharon R
authors.initials: SR
authors.lastname: Lewin
authors.forename: Paul R
authors.initials: PR
authors.lastname: Gorry
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Cherry
mesh_terms: D017209:Apoptosis; D004247:DNA / Q000737:chemistry; D053938:DNA Fragmentation; D015658:HIV Infections / Q000473:pathology; D006801:Humans; D019169:Jurkat Cells; D008970:Molecular Weight; D009707:Nucleosomes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D009707:Nucleosomes; D004247:DNA