ABSTRACT
Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.
Subject(s)
DNA/analysis , Fluorescent Dyes/chemistry , Multiplex Polymerase Chain Reaction/methods , DNA/metabolism , Fluorescent Dyes/metabolism , Humans , Protein Binding/physiology , Real-Time Polymerase Chain Reaction/methodsABSTRACT
DNA damage-induced cell-cycle "checkpoint" responses reduce the mutagenic effects of this damage. However, the maintenance of genomic stability comes at a price: the slowing of growth and a delay in the development of critical tissues. In mammals, every mutated cell has the potential to become cancerous and therefore lethal. In plants, the risk of lethal cancers is essentially nil and the costs of delays in development are very high. Here, we investigate DNA damage checkpoint responses in meristematic (root and shoot tip) versus strictly somatic (stomatal and endoreduplicating) tissues in plants. We find that the ionizing radiation (IR)-induced cell-cycle responses observed in the root and shoot tip meristems do not apply to more differentiated tissues.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Cell Cycle/physiology , DNA Damage/physiology , Seedlings/physiology , Arabidopsis/genetics , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Cotyledon/cytology , Cotyledon/physiology , Cotyledon/radiation effects , DNA Ligase ATP , DNA Ligases/genetics , DNA Ligases/metabolism , DNA Repair/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Radiation , Endonucleases/genetics , Endonucleases/metabolism , Gamma Rays , Hypocotyl/cytology , Hypocotyl/physiology , Hypocotyl/radiation effects , Meristem/cytology , Meristem/physiology , Meristem/radiation effects , Mutation , Organ Specificity , Seedlings/genetics , Seedlings/radiation effectsABSTRACT
The variant histone H2AX is phosphorylated in response to UV irradiation of primary human fibroblasts in a complex fashion that is radically different from that commonly reported after DNA double-strand breaks. H2AX phosphorylation after exposure to ionizing radiation produces foci, which are detectable by immunofluorescence microscopy and have been adopted as clear and consistent quantitative markers for DNA double-strand breaks. Here we show that in contrast to ionizing radiation, UV irradiation mainly induces H2AX phosphorylation as a diffuse, even, pan-nuclear staining. UV induced pan-nuclear phosphorylation of H2AX is present in all phases of the cell cycle and is highest in S phase. H2AX phosphorylation in G(1) cells depends on nucleotide excision repair factors that may expose the S-139 site to kinase activity, is not due to DNA double-strand breaks, and plays a larger role in UV-induced signal transduction than previously realized.