Construction of Strand-seq libraries in open nanoliter arrays

Single-cell Strand-seq generates directional genomic information to study DNA repair, assemble genomes, and map structural variation onto chromosome-length haplotypes. We report a nanoliter-volume, one-pot (OP) Strand-seq library preparation protocol in which reagents are added cumulatively, DNA purification steps are avoided, and enzymes are inactivated with a thermolabile protease. OP-Strand-seq libraries capture 10%-25% of the genome from a single-cell with reduced costs and increased throughput

Energy spread of ultracold electron bunches extracted from a laser cooled gas

Ultrashort and ultracold electron bunches created by near-threshold femtosecond photoionization of a laser-cooled gas hold great promise for single-shot ultrafast diffraction experiments. In previous publications the transverse beam quality and the bunch length have been determined. Here the longitudinal energy spread of the generated bunches is measured for the first time, using a specially developed Wien filter. The Wien filter has been calibrated by determining the average deflection of the electron bunch as a function of magnetic field. The measured relative energy spread $\frac{\sigma_{U}}{U} = 0.64 \pm 0.09\%$ agrees well with the theoretical model which states that it is governed by the width of the ionization laser and the acceleration length

Efficient assembly of very short oligonucleotides using T4 DNA Ligase

<p>Abstract</p> <p>Background</p> <p>In principle, a pre-constructed library of all possible short oligonucleotides could be used to construct many distinct gene sequences. In order to assess the feasibility of such an approach, we characterized T4 DNA Ligase activity on short oligonucleotide substrates and defined conditions suitable for assembly of a plurality of oligonucleotides.</p> <p>Findings</p> <p>Ligation by T4 DNA Ligase was found to be dependent on the formation of a double stranded DNA duplex of at least five base pairs surrounding the site of ligation. However, ligations could be performed effectively with overhangs smaller than five base pairs and oligonucleotides as small as octamers, in the presence of a second, complementary oligonucleotide. We demonstrate the feasibility of simultaneous oligonucleotide phosphorylation and ligation and, as a proof of principle for DNA synthesis through the assembly of short oligonucleotides, we performed a hierarchical ligation procedure whereby octamers were combined to construct a target 128-bp segment of the beta-actin gene.</p> <p>Conclusions</p> <p>Oligonucleotides as short as 8 nucleotides can be efficiently assembled using T4 DNA Ligase. Thus, the construction of synthetic genes, without the need for custom oligonucleotide synthesis, appears feasible.</p

Loss of CIC promotes mitotic dysregulation and chromosome segregation defects

Background: CIC is a transcriptional repressor inactivated by loss-of-function mutations in several cancer types, including gliomas, lung cancers, and gastric adenocarcinomas. CIC alterations and/or loss of CIC activity have been associated with poorer outcomes and more aggressive phenotypes across cancer types, which is consistent with the notion that CIC functions as a tumour suppressor across a wide range of contexts. Results: Using mammalian cells lacking functional CIC, we found that CIC deficiency was associated with chromosome segregation (CS) defects, resulting in chromosomal instability and aneuploidy. These CS defects were associated with transcriptional dysregulation of spindle assembly checkpoint and cell cycle regulators. We also identified novel CIC interacting proteins, including core members of the SWI/SNF complex, and showed that they cooperatively regulated the expression of genes involved in cell cycle regulation. Finally, we showed that loss of CIC and ARID1A cooperatively increased CS defects and reduced cell viability. Conclusions: Our study ascribes a novel role to CIC as an important regulator of the cell cycle and demonstrates that loss of CIC can lead to chromosomal instability and aneuploidy in human and murine cells through defects in CS, providing insight into the underlying mechanisms of CIC's increasingly apparent role as a "pan-cancer" tumour suppressor

Loss of CIC promotes mitotic dysregulation and chromosome segregation defects

Background: CIC is a transcriptional repressor inactivated by loss-of-function mutations in several cancer types, including gliomas, lung cancers, and gastric adenocarcinomas. CIC alterations and/or loss of CIC activity have been associated with poorer outcomes and more aggressive phenotypes across cancer types, which is consistent with the notion that CIC functions as a tumour suppressor across a wide range of contexts. Results: Using mammalian cells lacking functional CIC, we found that CIC deficiency was associated with chromosome segregation (CS) defects, resulting in chromosomal instability and aneuploidy. These CS defects were associated with transcriptional dysregulation of spindle assembly checkpoint and cell cycle regulators. We also identified novel CIC interacting proteins, including core members of the SWI/SNF complex, and showed that they cooperatively regulated the expression of genes involved in cell cycle regulation. Finally, we showed that loss of CIC and ARID1A cooperatively increased CS defects and reduced cell viability. Conclusions: Our study ascribes a novel role to CIC as an important regulator of the cell cycle and demonstrates that loss of CIC can lead to chromosomal instability and aneuploidy in human and murine cells through defects in CS, providing insight into the underlying mechanisms of CIC's increasingly apparent role as a "pan-cancer" tumour suppressor

Comprehensive molecular portraits of human breast tumours

This Article from the Cancer Genome Atlas consortium describes a multifaceted analysis of primary breast cancers in 825 people. Exome sequencing, copy number variation, DNA methylation, messenger RNA arrays, microRNA sequencing and proteomic analyses were performed and integrated to shed light on breast-cancer heterogeneity. Just three genes — TP53, PIK3CA and GATA3 — are mutated at greater than 10% frequency across all breast cancers. Many subtype-associated and novel mutations were identified, as well as two breast-cancer subgroups with specific signalling-pathway signatures. The analyses also suggest that much of the clinically observable plasticity and heterogeneity occurs within, and not across, the major subtypes of breast cancer

Contaminant-induced current decline in capillary array electrophoresis

This research clarifies, for the first time, the mechanism and impact of current decline in capillary array electrophoresis (CAE). High throughput capillary array electrophoresis instruments for DNA sequencing suffer to varying degrees from failure associated with electrophoretic current decline and inhibition or delay in the arrival of fragments at the detector. This effect is known to be associated with residual quantities of large, slow moving fragments of template or genomic DNA carried through from sample preparation and sequencing reactions. Here, we document and investigate the existence of an expanding ionic depletion region induced by overloading the capillary with low-mobility DNA fragments, and the effect of growth of this region on electrophoresis run failure. This depletion region forms upstream of the smaller sequencing fragments, and its expansion was found not to affect the quality of the sequencing peaks at the detector. Rather the current decline associated with depletion region growth reduces the velocity of the downstream sequencing fragments, so fewer fragments arrive at the detector during the run. It is shown, through analytical and numerical models, how increasing quantities of slow moving DNA cause the concentration of background electrolyte downstream to decline. With the concentration of such fragments beyond a threshold quantity, the anode-side boundary of the nascent depletion region is shown to propagate toward the anode at a rate faster than the contaminant DNA migration. Under such conditions the depletion region expands, the current declines, and the electrophoresis run suffers from a reduced yield of sequence data or fails completely. While the upstream boundary of the depletion region propagates with the DNA, the propagation rate of the downstream boundary is found to be inversely proportional to the amount of ionic depletion, and independent of the motion of the DNA. Observations suggest that these downstream boundaries may propagate as a result of an imbalance in current carriers brought about by the exposure of bound charges in the matrix or capillary wall, which may be coupled to a rise in pH.Science, Faculty ofPhysics and Astronomy, Department ofGraduat

A combined scanning tunneling microscope and scanning electron microscope

A scanning tunneling microscope (STM) was developed to work in conjunction with a Hitachi S-4100 field emission scanning electron microscope (SEM). To achieve the necessary five degrees of freedom for sample and probe movement, an entirely mechanical method was used, employing pairs of parallelogram flexure hinges actuated by set screws driven through gear reduction. The STM was also configured such that the sample is mounted on the piezoelectric scanner and the probe is fixed. This allowed the sample to be positioned close to the objective aperture of the SEM. The role of the SEM was envisioned primarily as an alignment tool to position the STM probe over a region of interest on the sample. The STM would then complement the SEM by revealing different surface detail. The instrument was successfully used this way, to image small structures such as Molecular Beam Epitaxy (MBE) and Metal Oxide Chemical Vapor Deposition (MOCVD) grown GaAs/AlGaAs heterostructures and long gate MOSFETs. It was also used to measure the depth of e-beam fabricated calibration pits. Two methods of STM lithography were investigated. Field evaporation of probe material produced mixed results, due in part to the experiments being done in vacuum. Field evaporation proved a useful method for clearing contamination from the probe. STM probe induced modification of passivated silicon surfaces was also investigated. A voltage threshold for depassivation was found and a model for formation of etch masks at positive polarity was proposed. Patterns were written and successfully transferred to the substrate by wet chemical etch. Lines were as thin as 20 nm, and up to 15 nm high. An important result of the lithography research was the discovery that the SEM could image depassivated regions at low accelerating voltages. This allowed some light to be cast on a number of STM imaging artifacts, as well as allowing precise calibration of imaging range. Artifacts included the effect of a finite tip radius on imaging steps and grooves, imaging on highly contaminated surfaces, multiple tips and dielectric material acquired by the probe near the tunneling junction. The effect of electron beam induced carbon deposition was investigated. The STM was used to measure the depth of thin carbon deposits. A deposition rate of between .6 and 2 nm/sec at 300,000X was found.Science, Faculty ofPhysics and Astronomy, Department ofGraduat