Research articles

To determine long-range linkage between single-nucleotide polymorphisms (SNPs) and the repeat-containing region of a disease-related gene, Liu et al. develop SNP linkage by circularization (SLiC) and lay the groundwork for using allele-specific RNA interference to target insertion or deletion mutations in disease-associated genes.

The two major mechanisms for peptide fragmentation by mass spectrometry, collision-activated dissociation (CAD) or a newer method, electron transfer dissociation (ETD), display different efficacies for different peptide chemistries. A decision tree algorithm, which can be embedded into instruments with both CAD and ETD capabilities, selects the optimal fragmentation method to improve the chances of successful peptide identification.

Directed evolution experiments usually rely on high-throughput screening of very large libraries of mutants, but most of the mutants do not even yield stable, functional proteins. The concept of neutral drift can be used to generate small but highly polymorphic and stable mutant libraries as a starting point for further evolution.

The combination of single-molecule fluorescence resonance energy transfer measurements of multiple dye pairs with probabilistic data analysis allows quantitative measurement of the position of flexible domains in macro-molecular complexes. The method was used to determine the three-dimensional probability density of the position of RNA exiting the transcription elongation complex.

A miniature epifluorescence microscope that can be carried by a freely-moving adult mouse allows cellular-level imaging of neuronal spiking or measurement of microcirculation during normal behavioral activities.

A strategy using 48 or more singly labeled fluorescent oligonucleotide probes targeted to individual mRNA molecules allows the simultaneous localization and quantification of three mRNA species in fixed cells. mRNA visualization in whole animals and other organisms is also demonstrated.

Time-resolved wide-angle X-ray scattering (TR-WAXS) using synchrotron radiation can be used to observe dynamic protein structural changes with nanosecond time resolution in solution, complementing time-resolved optical spectroscopy and Laue crystallography methods.

Spectral searching, based on matching experimental peptide spectra to reference spectral libraries, is gaining interest as an alternative to traditional sequence-database searching in mass spectrometry–based proteomics. A software tool, SpectraST, now allows users to build their own high-quality spectral libraries from raw data.

A simple yet powerful super-resolution imaging approach based on switching off ordinary fluorophores and localizing those remaining or regaining fluorescence is illustrated using continuous widefield illumination and imaging of fixed and living cells labeled with rhodamine-derived dyes or fluorescent proteins. Biteen et al., also in this issue, describe related work using the ordinary fluorophore of EYFP for super-resolution imaging.

Previous work showed that the commonly used fluorescent protein EYFP can be bleached and reactivated. Exploiting this property allows super-resolution in vivo imaging of EYFP-labeled structures in living bacteria. Fölling et al., also in this issue, describe a related approach for super-resolution imaging using other ordinary fluorophores.

Targeted regions of the human genome are resequenced in multiplex with Illumina technology, and the pipeline is evaluated for polymorphism discovery and genotyping.

Using both behavioral and electrophysiological readouts, Channelrhodopsin-2, a light-gated cation channel, is applied to the study of synaptic function in Caenorhabditis elegans.

An automated sorting method using the COPAS Biosort machine allows the isolation of mutant C. elegans displaying differences in GFP expression in small numbers of cells. Compared to manual methods this increases the efficiency of the phenotypic selection step in cell-fate screens.

A simplified strategy to enzymatically preadenylate bar-coded oligonucleotides to be used for capturing microRNAs in biological samples is described. This efficient method should greatly facilitate multiplex analysis and profiling of microRNAs.

A chromatin immunoprecipitation and sequencing (ChIP-Seq) data analysis package, QuEST, facilitates transcription factor binding site discovery at about 20-base-pair resolution.

A new prediction algorithm for microRNA targets, mirWIP, is presented. The algorithm weights target site features based on their enrichment in an experimentally defined immunoprecipitation dataset and identifies verified miRNA-mRNA interactions in Caenorhabditis elegans with improved specificity compared to current methods.

To study long-term changes in neuronal circuits at single-cell resolution, a Troponin C–based Ca²⁺ indicator protein has been reengineered to increase the signal strength. This allows repeated measurements, over days and weeks, of orientation selective neurons in mouse visual cortex. Hasan et al., also in this issue, describe the use of a similar sensor for recording neuronal activity in vivo.

Measurement of in vivo neuronal activity with single neuron and single action potential resolution is important for studying neuronal function. Delivery of a FRET-based fluorescent Ca²⁺ indicator protein using adeno-associated virus results in high expression levels allowing in vivo detection of single action potentials at low firing rates. Griesbeck et al., also in this issue, describe the use of a similar sensor for recording neuronal activity in vivo.

Holographic illumination allows the production of complex, user-defined, two-dimensional illumination patterns. Used to manipulate light-sensitive molecules in cells, this system permits their simultaneous excitation at multiple locations of arbitrary shape and size—facilitating spatial and temporal regulation of cell function.

Identifying the molecular lesions in mutants isolated in forward genetic screens can be a laborious process. A proof-of-principle study in Caenorhabditis elegans now shows that this can be achieved rapidly by whole-genome deep sequencing.