Brockladefoged2544

The local mechanical properties of the DNA polymer influence molecular processes in biology that require mechanical deformations of DNA. Lack of suitable high-throughput experimental techniques had precluded measuring how these properties might vary with sequence along the vast lengths of genomes. Here, we present a detailed protocol for a recently developed experimental technique called loop-seq, which measures at least one local mechanical property of DNA-its propensity to cyclize-in genome-scale throughput. Loop-seq has been used to obtain experimentally derived genome-wide maps of a physical property of DNA. Such measurements have revealed that diverse DNA-deforming processes involved in chromatin organization at various genomic loci are regulated by the genetically encoded, sequence-dependent variations in the mechanical properties of DNA.Under normal conditions, the genome of eukaryotic cells is faithfully replicated during S phase. However, in cells exposed to DNA polymerase inhibitors, some regions of the genome may fail to be replicated prior to mitotic entry. To prevent chromosomal breakage and loss of genomic information, mitotic DNA synthesis (MiDAS) completes replication of the genome prior to the onset of anaphase. We have developed a protocol that allows one to map the genomic regions that are replicated by MiDAS in mammalian cells. The protocol involves incorporation of a thymidine analog in nascent DNA in mitotic cells and then capture and high throughput sequencing of the nascent DNA. With this approach, sites of MiDAS can be identified at high resolution.Variations in the genetic information originate from errors during DNA replication, error-prone repair of DNA damages, or genome editing. The most common approach to detect changes in DNA sequences employs sequencing technologies. However, they remain expensive and time-consuming, limiting their utility for routine laboratory experiments. We recently developed DinucleoTidE Signature CapTure (DTECT). DTECT is a marker-free and versatile detection method that captures targeted dinucleotide signatures resulting from the digestion of genomic amplicons by the type IIS restriction enzyme AcuI. Here, we describe the DTECT protocol to identify mutations introduced by CRISPR-based precision genome editing technologies or resulting from genetic variation. DTECT enables accurate detection of mutations using basic laboratory equipment and off-the-shelf reagents with qualitative or quantitative capture of signatures.DNA double-strand breaks in DNA (DSBs) are common yet highly detrimental events in living organisms. To repair the damage, each cell requires a coordinated set of DNA damage response (DDR) proteins that can respond quickly, effectively, and precisely. Better understanding of these processes is therefore essential and would require an effective means of inducing targeted DSBs on demand, but previous methods are hampered by limited control over genomic location, timing, or lesion types. Tight spatiotemporal control of CRISPR-Cas9 activity has potential to overcome these limitations, which led to the development of two methods for rapid activation or deactivation of Cas9 using light. In this chapter, we discuss how control of Cas9 can advance DDR studies, describe protocols to control Cas9 activation and deactivation using this new technology, and finally outline three compatible readouts of DNA damage and the cellular response DSB levels using droplet digital PCR, repair factor localization using ChIP-seq, and insertion-deletion (indel) repair outcomes using Sanger sequencing.Endonucleolytic cleavage of DNA ends by the human Mre11-Rad50-Nbs1 (MRN) complex occurs in a manner that is promoted by DNA-dependent Protein Kinase (DNA-PK). A method is described to isolate DNA-PK-bound fragments released from chromatin in human cells using a modified Gentle Lysis and Size Selection chromatin immunoprecipitation (GLASS-ChIP) protocol. This method, combined with real-time PCR or next-generation sequencing, can identify sites of MRN endonucleolytic cutting adjacent to DNA-PK binding sites in human cells.Mitotic crossovers have the potential to cause large-scale genome rearrangements. Here, we describe high-throughput, single-cell, whole-genome sequencing methods for mapping crossovers genome-wide at scale. The methods are generalizable to various eukaryotes and to other end points requiring high-throughput, high-coverage single cell sequencing.Diverse DNA structures occur as reaction intermediates in various DNA-damage and -repair mechanisms, most of which results from replication stress. We harness the power of proteins evolutionarily optimized to bind and "trap" specific DNA reaction-intermediate structures, to quantify the structures, and discern the mechanisms of their occurrence in cells. The engineered proteins also allow genomic mapping of sites at which specific DNA structures occur preferentially, using a structure-trapping protein and ChIP-seq- or Cut-and-Tag-like methods. Genome-wide identification of sites with recurrent DNA-damage intermediates has illuminated mechanisms implicated in genome instability, replication stress, and chromosome fragility. Here, we describe X-seq, for identifying sites of recurrent four-way DNA junctions or Holliday-junctions (HJs). X-seq uses an engineered, catalysis-defective mutant of Escherichia coli RuvC HJ-specific endonuclease, RuvCDefGFP. X-seq signal indicates sites of recombinational DNA repair or replication-fork stalling and reversal. We also describe methods for genomic mapping of 3'-single-stranded DNA ends with SsEND-seq, in E. coli. Both methods allow genomic profiling of DNA-damage and -repair intermediates, which can precede genome instability, and are expected to have many additional applications including in other cells and organisms.A detailed understanding of how homologous recombination proceeds at the molecular level in vivo requires the ability to detect in real time the appearance of specific intermediates of DNA repair. The most detailed analysis of double-strand break (DSB) repair in eukaryotes has come from the study of budding yeast, using an inducible site-specific HO endonuclease to initiate recombination synchronously in nearly all cells of the population. Polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) methods have been used to visualize the timing of the DSB, its resection by 5' to 3' exonucleases, the binding of the Rad51 recombinase and the pairing of the Rad51 filament with a homologous donor sequence. PCR has also been used to identify the next key step the initiation of new DNA synthesis to extend the invading stand and copy the donor template. In break-induced replication (BIR), there appears to be a very long delay between strand invasion and this primer extension step. Here we describe an alternative method, an assay for monitoring BIR elongation rate (AMBER) based on digital droplet PCR that yields a much earlier time of initial DNA synthesis. We suggest that previous methods have failed to recover the initial long, single-stranded primer extension product that is readily detected by AMBER.The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem. In particular, the use of site-specific nucleases capable of inducing DNA nicks that replication converts into double-strand breaks has enabled the specific study of the repair of such replication-born double strand breaks by sister chromatid recombination. In this chapter, we describe detailed protocols for determining the efficiency and kinetics of this recombination reaction as well as for the genetic quantification of recombination products.The in vitro reconstitution of origin firing was a key step toward the biochemical reconstitution of eukaryotic DNA replication in budding yeast. Today the basic replication assay involves proteins purified from 24 separate protocols that have evolved since their first publication, and as a result, the efficiency and reliability of the in vitro replication system has improved. Here we will present protocols for all 24 purifications together with a general protocol for the in vitro replication assay and some tips for troubleshooting problems with the assay.Core stability exercises and exercises that stimulate sensory-motor information are recommended for the prevention of injuries and the maintenance and rehabilitation of deficits related to postural control (PC). GDC-0077 However, the comparison of results between core stability and sensory-motor exercises in the literature is limited to sitting and standing positions.

To determine the acute effect of core stability and sensory-motor exercises on PC during sitting and standing in young adults.

A total of 39 participants, with a mean age of 23 years, were randomly divided into three groups (1) Core stability exercises; (2) Sensory-motor exercises; (3) Control. Each group performed a sequence of five specific exercises of core stability and sensory-motor exercises (except controls). PC was evaluated before and after exercise in the seated and the one-legged stance conditions using a force platform.

No significant difference was found for any variables of postural oscillation (P>0.05) among the three groups studied. The magnitude of the effect of interventions in general was a small to moderate effect (d=0.02/-0.48).

The findings show that acute intervention with core stability and sensory-motor exercises did not produce any significant effects (reduction of postural oscillation) on PC during sitting and standing positions in young adults.

The findings show that acute intervention with core stability and sensory-motor exercises did not produce any significant effects (reduction of postural oscillation) on PC during sitting and standing positions in young adults.Indirect techniques of predicting hand grip force are fundamental to develop hand control systems for assistive devices. The purpose of this study was to determine the reliability of 3D position of forearm surface at different isometric hand grip forces. Three-dimensional motion analysis was used to measure displacement of 24 discrete and standardized surface markers placed on forearm in 20 healthy participants. The relative displacements were measured for isometric grip forces at 0%, 5%, 20% 50% and 80% of maximum voluntary contraction (MVC). Intraclass correlation coefficient (ICC) for relative radial displacement (RRD) of each marker was calculated. Averaged single measure ICC of 24 markers at five grip forces was 0.61; while the highest averaged single measure ICC (0.80) for all markers was achieved at 80% of MVC and the lowest (0.47) at 0% of MVC. The average measure ICC for each grip force across the 24 markers also increased with grip force from 0.80 at 0% of MVC to the maximum of 0.95 at 80% of MVC. In conclusion, RRD showed moderate and high ICCs for single and average measures respectively.