We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains a mismatched nucleotide or a deletion. The probes are particularly suited for monitoring the synthesis of specific nucleic acids in real time. When used in nucleic acid amplification assays, gene detection is homogeneous and sensitive, and can be carried out in a sealed tube. When introduced into living cells, these probes should enable the origin, movement, and fate of specific mRNAs to be traced.
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"The Molecular beacon (MB) usually is constructed with 25-35 nucleotides and a specific stemloop conformation that allow the maintenance of the fluorescence by resonance energy transfer (RET) (Tyagi et al. 1996). When the MB hybridizes with the target, there is a change in the loop conformation, resulting in an increase of the distance between the fluorophore and the quencher, breaking the FRET, allowing the fluorescence detection (Tyagi et al. 1996) The MB technology applied to environmental virology is still recent and limited, but this method was reported for virus detection, showing an specific binding on viral DNA in clinical samples (Dunams et al. 2012). "
[Show abstract][Hide abstract] ABSTRACT: Gastroenteritis, diarrhea, and other diseases can be caused by enteric viruses transmitted by fecal-oral route. Human adenoviruses (HAdV), rotavirus A and C (RVA and RVC, respectively), hepatitis A and E virus (HAV and HEV, respectively), human astroviruses (HAstV), human noroviruses (HuNV) and enteroviruses (EV) are, among the enteric viruses, the most frequently detected in environment samples. These viruses are usually introduced into aquatic environments by human, industrial, or agricultural activities and are widely distributed all over the world. They have the common characteristics to be structurally stable and can also absorb to solid particles and biofilms, thereby protecting themselves from inactivating factors. This revision aimed to present and discuss: i) most relevant enteric viruses for human and animal health; ii) enteric viruses as contaminants and bioindicators in environmental samples; iii) molecular and cell culture methods for enteric virus detection; iv) use of enteric viruses for microbial risk assessment. Impacts of enteric viruses on environment and the potential use as bioindicators of the sanitary security, such as presence and infectivity studies were discussed as development of new tools for disinfection, monitoring, risk modeling and management, among other studies.
"The quencher can be coupled to a second complementary oligonucleotide, which is displaced upon binding to the target sequence (Morrison et al., 1989). In so-called molecular beacons, the quencher is coupled to the same oligonucleotide (Tyagi and Kramer, 1996; Vargas et al., 2005). Molecular beacons contain small complementary sequences on both ends, thereby forcing the unbound molecule into a hairpin configuration, in which the dye and the quencher are kept in close proximity. "
[Show abstract][Hide abstract] ABSTRACT: RNA molecules carry out widely diverse functions in numerous different physiological processes in living cells. The RNA lifecycle from transcription through processing of nascent RNA to the regulatory function of non-coding RNA and cytoplasmic translation of messenger RNA has been studied extensively using biochemical and molecular biology techniques. In this Commentary, we highlight how single molecule imaging and particle tracking can yield further insight into the dynamics of RNA particles in living cells. In the past few years, a variety of bright and photo-stable labelling techniques have been developed to generate sufficient contrast for imaging of single endogenous RNAs in vivo. New imaging modalities allow determining not only lateral but also axial positions with high precision within the cellular context and across a wide range of specimen from yeast and bacteria to cultured cells and even multicellular organisms or live animals. A whole range of methods to localize and track single particles and analyse trajectory data are available to yield detailed information about the kinetics of all parts of the RNA lifecycle. While the concepts presented are applicable to all types of RNA, we showcase here the wealth of information gained from in vivo imaging of single particles by discussing studies investigating dynamics of intranuclear trafficking, nuclear pore transport and cytoplasmic transport of endogenous messenger RNA.
No preview · Article · Oct 2015 · Journal of Cell Science
"Single-cell transcriptomics started with the development of single-cell qPCR (Bengtsson et al., 2008; Eberwine et al., 1992; Huang et al., 2014; Taniguchi et al., 2009; Warren et al., 2006), which, alongside single-molecule FISH, is still the method of choice for targeted analysis of transcripts (Femino et al., 1998; Raj et al., 2006, 2008; Tyagi and Kramer, 1996). Based on advances made in the single-cell qPCR field, whole-transcriptome analysis was performed, using microarrays (Kamme et al., 2003; Kurimoto et al., 2006, 2007; Subkhankulova et al., 2008). "