A novel and promising method was developed to extract mRNA from a single cell based on the functionalized superparamagnetic nanoparticles. The oligo(dT)‐coupled magnetite nanobeads were synthesized by the reaction of oligo(dT) and thiol‐modified γ‐Fe2O3 nanoparticles. The single cell was isolated from the massive cultivation according to a semi‐quantum approaching technique and then lysed before mRNA separation. The oligo(dT)‐coupled magnetite nanobeads were added to the crude lysates and then magnetic separation was preformed to get mRNA. The mRNA amplification through a two‐step RT‐PCR method was achieved. The agarose gel electrophoresis of PCR products after amplification shows that mRNA could be extracted from a single cell successfully. 相似文献
We use spectrally‐resolved room temperature single molecule spectroscopy to yield insights into the occurrence and dynamics of spectral forms of single tetramers of DsRed and its variants DsRed2, Fluorescent Timer, DsRed_N42H and AG4. The red‐emitting chromophore in DsRed and all studied variants readily converts into a high quantum efficiency super‐red emitting form. We propose the existence of two super‐red forms of different quantum efficiencies. The observed emission from the green‐emitting chromophore is consistent with bulk spectroscopy. We further observe distinct new spectral forms from each variant, which we attribute to a photoinduced chemical reaction leading to a truncated form of the red‐emitting chromophore analogous to the chromophore in the visible fluorescent protein zFP538. Our results have implications for the accurate interpretation of biological and biochemical processes illuminated by fluorescent proteins as well as for choosing appropriate experimental configurations. 相似文献
Singled out for its singularity : In a single‐step, single‐component, fluorescence‐based method for the detection of single‐nucleotide polymorphisms at room temperature, the sensor is comprised of a single, self‐complementary DNA strand that forms a triple‐stem structure. The large conformational change that occurs upon binding to perfectly matched (PM) targets results in a significant increase in fluorescence (see picture; F=fluorophore, Q=quencher).
This paper presents a systematic method to isolate and trap long single DNA segments between integrated electrodes in a microfluidic environment. Double stranded lambda-DNA molecules are introduced in a microchip and are isolated by electrophoretic force through microfluidic channels. Downstream, each individual molecule is extended and oriented by ac dielectrophoresis (900 kHz, 1 MV m(-1)) and anchored between aluminium electrodes. With a proper design, a long DNA segment (up to 10 microm) can be instantly captured in stretched conformation, opening way for further assays. 相似文献
Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli‐DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli‐BI complexes in cells. Broccoli‐BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis‐BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers. 相似文献
To each his own : An addressable electrochemical device consisting of orthogonally arranged rows and columns of electrodes has been constructed to monitor protein expression in genetically engineered cells at the single‐cell level. The response based on redox cycling reflected the different expression levels of the enzyme from individual HeLa cells transfected with a plasmid vector including secreted alkaline phosphatase.
Spectral diffusion as a result of both the transitions between different molecular conformers and the ′′molecular softness′′ of quasi‐free perylene diimides on a SiO2 surface is investigated by means of single‐molecule spectroscopy, which reveals the time dependence of both the fluorescence spectra and the three‐dimensional orientation. Spectral wavelengths of all single emitters cover a wide energy range of about 0.27 eV, which is due to different types of conformers with large differences in optical transition energy. Time‐dependent spectral trajectories of single emitters within this wavelength manifold are evaluated with a model transcribed from the analysis of spatial diffusion. Spectral diffusion processes are closely correlated with fluorescence emission and excitation power. The overall analysis of spectral diffusion reveals, similar to proteins, a hierarchy of energy barriers in a broad energy landscape. 相似文献