Specific DNA Segments Research Articles

Novel Electrochemical Sensor Based on One-Step Encapsulation of Metal-Organic Framework (MOF) for Simultaneous Detection of SARS-CoV and SARS-CoV-2.

This study investigates three metal-organic frameworks (MOFs) with distinct Brunauer-Emmett-Teller (BET) surface areas and pore sizes: MOFZr (1274 m2/g, <0.7 nm), MOFAl (371 m2/g, 1.5 nm), and MOFCr (1917 m2/g, 2 nm). Methylene blue (MB, 1.4 × 0.7 × 0.2 nm3) and triferrocene (tri-FC, 1.2 × 0.9 × 0.3 nm3) were adsorbed onto these MOFs. Specific DNA segments targeting SARS-CoV-2 (PR and PO) were employed to block the pores of the MOFs, leading to the formation of DNA-gated MOFs: MOFX/MB/PR and MOFX/tri-FC/PO. These constructs were subsequently integrated with four-arm poly(ethylene glycol) amine (4-arm-PEG-NH2)-modified gold electrodes to create the corresponding sensors (MOFX/MB/PR+MOFX/tri-FC/PO@4-arm-PEG-NH2@Au NPs@GCE). The DNA-gated MOFAl system exhibited the highest loading capacity and a 3-fold increase in sensing efficiency following the introduction of SARS-CoV-2 target DNA, surpassing the performance of the other systems. This study highlights the enhancement of the DNA-gated MOFs when the three-dimensional structures of the guest molecules closely align with the pore sizes of the MOFs. It emphasizes a critical aspect of the traditional design approach for electrochemical sensors based on MOF encapsulation, which often prioritizes BET surface areas while neglecting the compatibility between the sizes of guest molecules and MOF pore diameters. Moreover, the sensor effectively discriminated between SARS-CoV-2 and SARS-CoV by precisely aligning the SARS-CoV-2 target DNA with PR and PO. In contrast, the specific genetic targets (SR) from SARS-CoV showed complete mismatches with PO and a three-base deviation with PR. This differentiation was achieved in a simple one-step assay, even in 5% serum, across a linear concentration range of 10-9 to 10-14 M. This range signifies an expansion of 2 to 3 orders of magnitude compared to sensors that inaccurately selected MOFs.

A bivariate fluorescence biosensor based on Janus DNA nanoarchitecture-loaded dual-emissive Ag nanoclusters as bi-responsive signaling reporters

Constructing label-free bivariate fluorescence biosensor would be intriguing and desired for the recognizable and accurate detection of two specific DNA segments, yet the design of functional DNA structures with low overlapped interference might be challenging. Herein in this work, a double-faced Janus DNA nanoarchitecture (JDNA) with bi-responsive recognition regions on opposite sides was assembled, which consisted of two substrate strands and two template strands for loading green-/red-emissive Ag nanoclusters (gAgNC and rAgNC) as bivariate signaling reporters. Of note, the hybridized double helix in the middle rationally oriented two flank faces and stabilized the rigid conformation of JDNA, while the template sequences of bicolor clusters were blocked to minimize non-specific background leakage. Upon inputting two targets, the discernible hairpins lost their hairpin structures due to forming two dsDNA complexes. They were executed to simultaneously invade JDNA for activating two individual target-recycled strand displacement (TRSD) events, guiding signal transduction and efficient amplification. Consequently, the clustering templates were unlocked via the tailored conformation switch of JDNA, in which gAgNC and rAgNC were in situ synthesized in two diagonal positions, thereby significantly emitting bi-responsive signal without cross interference. Benefited from the logic integration of double-faced JDNA and TRSD, a label-free, sensitive and specific bivariate fluorescence approach was developed, which would open a new avenue for the potential application in biosensing and bioanalysis.

DNA barcoding, phylogeography and evolutionary dynamics of Chrysichthys auratus

This study embarked on an exploration into the genetic structure and evolutionary history of the Chrysichthys auratus species, leveraging PCR amplification, phylogenetic trees, and haplotype networks. Specific DNA segments were successfully amplified and visualized through electrophoresis. Newly obtained sequences were Bank into GenBank and given accession numbers (OR730807-OR730808-OR730809). The Neighbor-Joining method provided insights into the evolutionary relationships among taxa, further augmented by bootstrap values and the Tamura 3-parameter method. A comprehensive geographical haplotype network showcased pronounced genetic differentiation, especially between remote populations. Nonetheless, shared haplotypes between proximate regions indicated either ancestral genetic connections or ongoing gene flow. Employing the COI-DNA barcodes, an in-depth understanding of intra- and inter-populational genetic diversity was achieved. The study's findings unravel the intricate genetic landscape and evolutionary dynamics of C. auratus, offering novel perspectives into its demographic history across its vast native habitat.

Decoding the aging nexus: unravelling genetic networks and pharmacological strategies for lifespan extension and the methuselah paradox

We are all interested in knowing- whether genes and drugs can increase our life-span. As per Bible, Methuselah's lifespan lasted for a total of 969 years. Recent research has identified the Methuselah gene, a specific DNA segment that holds the potential to promote robust and healthy aging. This discovery opens new avenues for the development of pharmaceutical interventions aimed at extending human lifespan. Aging, a complex process influenced by natural selection, has evolved over time, adapting to factors such as cellular senescence and genetic instability. Research on aging has extensively employed invertebrate models like cnidarians, worms, flies, and yeast. Utilizing genetic methodologies with these organisms has resulted in the identification of numerous aging genes. Remarkably, there is compelling evidence of evolutionary conservation within longevity pathways across diverse species, including mammals. In search of omic study, we would consider data from another set of experiments performed on Cnidarians and show that there has a great advanced on the `biology of aging’ in an indirect way. Cnidarians, like Turritopsis dohrnii, showcase "ontogeny reversal," reverting to earlier stages, thus achieving biological immortality through repeated rejuvenation after reproduction. Alternatively, compounds like resveratrol and rapamycin, have been identified as having the ability to decelerate aging in model organisms. However, as of now, only rapamycin has demonstrated an impact on longevity in experiments on mice. The opportunity to postpone human aging currently exists, whether through established groups of tiny molecules or numerous emerging alternatives. In this context, we explore the approaches to convert findings from age-related research into pharmaceuticals.

In-house Extraction and Purification of Pfu-Sso7d, a High-processivity DNA Polymerase.

The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase. Key features • We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d. • Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance. • Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers. • The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCRapplications.

The association of positive sepsis PCRs tests with patient mortality, length of stay, and antibiotic management in a medical intensive care unit

Background: Polymerase chain reaction (PCR) testing amplifies a specific DNA segment through heat cycling to identify pathogens for the diagnosis of infection. This testing allows for rapid detection of pathogens, before conventional blood culture results become available. The high mortality associated with severe sepsis and septic shock stresses the importance of early diagnosis and initiation of early antibiotic therapy. This study analyzed the association of the sepsis PCR results and antibiotic management, length of stay, and mortality outcomes in a medical intensive care unit (MICU).&#x0D; Methods: This study is a retrospective, cross-sectional study on patients diagnosed with severe sepsis and septic shock based on ICD-10 codes, aged 18 years and older, admitted to the MICU at University Medical Center, Lubbock, Texas, between December 2016 to December 2020.&#x0D; Results: Clinical information from 268 patients with the diagnosis of sepsis or septic shock was collected and analyzed. The mean age was 60.9 ± 15.6 years with a predominance of men (144, 53.7%) and Caucasian race (193,73.4%). A total of 101 patients (37.6%) had positive PCR results; overall, PCR test results had a diagnostic sensitivity of 91.2 %. The concordance between positive blood culture and positive PCR was 93.3 % (p &lt;.0001). There was a significant correlation between PCR positivity and increased serum lactate levels (p=0.03), changes in antibiotics (P&lt;0.00010), and increased mortality rate (p=0.04). There was no significant correlation between PCR positivity and length of stay (p=0.84).&#x0D; Conclusion: In this study, PCR testing was an accurate tool for early identification of bacterial pathogens. A positive PCR was associated with higher serum lactate levels, higher mortality rates, and an increased frequency of antibiotic changes but was not associated with shorter ICU length of stay.&#x0D; Key words: Sepsis, septic shock, PCR testing, outcomes

Application of gene editing technology in Escherichia coli

Gene editing technology can be used to modify the genome of Escherichia coli for the investigation of gene functions, or to change the metabolic pathways for the efficient production of high-value products in engineered strains with genetic stability. A variety of gene editing technologies have been applied in prokaryotes, such as λ-Red homologous recombination and CRISPR/Cas9. As a traditional gene editing technique, λ-Red recombination is widely used. However, it has a few shortcomings, such as the limited integration efficiency by the integrated fragment size, the cumbersome gene editing process, and the FRT scar in the genome after recombination. CRISPR/Cas9 is widely used for genome editing at specific sites, which requires specific DNA segments according to the editing site. As the understanding of the two technologies deepens, a variety of composite gene editing techniques have been developed, such as the application of λ-Red homologous recombination in combination with homing endonucleaseⅠ-SceⅠ or CRISPR/Cas9. In this review, we summarized the basic principles of common gene editing techniques and composite gene editing techniques, as well as their applications in Escherichia coli, which can provide a basis for the selection of gene editing methods in prokaryotes.

Understanding the Emerging Link Between Circadian Rhythm, Nrf2 Pathway, and Breast Cancer to Overcome Drug Resistance.

The levels of different molecules in the cell are rhythmically cycled by the molecular clock present at the cellular level. The circadian rhythm is closely linked to the metabolic processes in the cells by an underlying mechanism whose intricacies need to be thoroughly investigated. Nevertheless, Nrf2 has been identified as an essential bridge between the circadian clock and cellular metabolism and is activated by the by-product of cellular metabolism like hydrogen peroxide. Once activated it binds to the specific DNA segments and increases the transcription of several genes that play a crucial role in the normal functioning of the cell. The central clock located in the suprachiasmatic nucleus of the anterior hypothalamus synchronizes the timekeeping in the peripheral tissues by integrating the light-dark input from the environment. Several studies have demonstrated the role of circadian rhythm as an effective tumor suppressor. Tumor development is triggered by the stimulation or disruption of signaling pathways at the cellular level as a result of the interaction between cells and environmental stimuli. Oxidative stress is one such external stimulus that disturbs the prooxidant/antioxidant equilibrium due to the loss of control over signaling pathways which destroy the bio-molecules. Altered Nrf2 expression and impaired redox balance are associated with various cancers suggesting that Nrf2 targeting may be used as a novel therapeutic approach for treating cancers. On the other hand, Nrf2 has also been shown to enhance the resistance of cancer cells to chemotherapeutic agents. We believe that maximum efficacy with minimum side effects for any particular therapy can be achieved if the treatment strategy regulates the circadian rhythm. In this review, we discuss the various molecular mechanisms interlinking the circadian rhythm with the Nrf2 pathway and contributing to breast cancer pathogenesis, we also talk about how these two pathways work in close association with the cell cycle which is another oscillatory system, and whether this interplay can be exploited to overcome drug resistance during chemotherapy.

In vivo diversification of target genomic sites using processive base deaminase fusions blocked by dCas9

In vivo mutagenesis systems accelerate directed protein evolution but often show restricted capabilities and deleterious off-site mutations on cells. To overcome these limitations, here we report an in vivo platform to diversify specific DNA segments based on protein fusions between various base deaminases (BD) and the T7 RNA polymerase (T7RNAP) that recognizes a cognate promoter oriented towards the target sequence. Transcriptional elongation of these fusions generates transitions C to T or A to G on both DNA strands and in long DNA segments. To delimit the boundaries of the diversified DNA, the catalytically dead Cas9 (dCas9) is tethered with custom-designed crRNAs as a “roadblock” for BD-T7RNAP elongation. Using this T7-targeted dCas9-limited in vivo mutagenesis (T7-DIVA) system, rapid molecular evolution of the antibiotic resistance gene TEM-1 is achieved. While the efficiency is demonstrated in E. coli, the system can be adapted to a variety of bacterial and eukaryotic hosts.

Urinary circulating DNA and circulating antigen for diagnosis of schistosomiasis mansoni: a field study.

To evaluate three non-invasive assays for the diagnosis of schistosomiasis mansoni in an Egyptian village. Urine was collected for the detection of circulating cathodic antigen (CCA) and cell-free parasite DNA (cfpd) by Point-of-contact (POC)-cassette assay and PCR, respectively. These tests were compared to Kato-Katz (KK) faecal thick smear for detection of Schistosoma mansoni eggs. Disease prevalence by POC-CCA assay was 86%; by PCR it was 39% vs. 27% by KK. Compared to KK, the sensitivity of POC-CCA reached 100%, but its specificity was only 19.2% with 41% accuracy. Sensitivity of the PCR assay for cfpd was 55.56%, and specificity was 67.12% with 64% accuracy. A new end point was calculated for combined analysis of KK, POC-CCA assay and PCR. Sensitivity for the three tests was 52.94%, 90.2% and 76.47%; specificity was 100% for KK and PCR and 18.37% for POC-CCA. The accuracy calculated for the three tests at the end point was 76% for KK, 55% for POC-CCA assay and 88% for PCR. Conventional PCR assay for detection of cfpd provides a potential screening tool for intestinal schistosomiasis with reliable specificity, reasonable accuracy and affordable financial and technical cost.

A Genome Model to Explain Major Features of Neurodevelopmental Disorders in Newborns.

The purpose of this study was to test the hypothesis that infections are linked to chromosomal anomalies that cause neurodevelopmental disorders. In children with disorders in the development of their nervous systems, chromosome anomalies known to cause these disorders were compared with foreign DNAs, including known teratogens. Genes essential for neurons, lymphatic drainage, immunity, circulation, angiogenesis, cell barriers, structure, epigenetic and chromatin modifications were all found close together in polyfunctional clusters that were deleted or rearranged in neurodevelopmental disorders. In some patients, epigenetic driver mutations also changed access to large chromosome segments. These changes account for immune, circulatory, and structural deficits that accompany neurologic deficits. Specific and repetitive human DNA encompassing large deletions matched infections and passed rigorous artifact tests. Deletions of up to millions of bases accompanied infection-matching sequences and caused massive changes in human homologies to foreign DNAs. In data from 3 independent studies of private, familial, and recurrent chromosomal rearrangements, massive changes in homologous microbiomes were found and may drive rearrangements and encourage pathogens. At least 1 chromosomal anomaly was found to consist of human DNA fragments with a gap that corresponded to a piece of integrated foreign DNA. Microbial DNAs that match repetitive or specific human DNA segments are thus proposed to interfere with the epigenome and highly active recombination during meiosis, driven by massive changes in human DNA-foreign DNA homologies. Abnormal recombination in gametes produces zygotes containing rare chromosome anomalies that cause neurologic disorders and nonneurologic signs. Neurodevelopmental disorders may be examples of assault on the human genome by foreign DNAs at a critical stage. Some infections may be more likely tolerated because they resemble human DNA segments. Even rare developmental disorders can be screened for homology to infections within altered epigenomes and chromatin structures. Considering effects of foreign DNAs can assist prenatal and genetic counseling, diagnosis, prevention, and early intervention.

Qualitative real-time PCR identification of the khapra beetle, Trogoderma granarium (Coleoptera: Dermestidae)

The identification of insect species is generally achieved by analyzing the specific morphological characteristics or, in more recent years, by polymerase chain reaction (PCR) assays of DNA sequences. PCR assays, which detect specific segments of DNA, have been established as a highly sensitive method for a wide range of applications, including the verification of hereditary diseases and for criminal investigations. For the identification of insects, the CO1 (cytochrome c oxidase subunit I) gene of mitochondrial (mt) DNA is often used. However, analyses of the entire mtDNA sequence might yield more reliable regions for use in the identification of insect species. Here we developed a method for real-time PCR identification of the khapra beetle, Trogoderma granarium Everts, by comparing the whole mtDNA sequences of three species of dermestid beetle: the khapra beetle, the varied carpet beetle, Anthrenus verbasci (L.), and the black carpet beetle, Attagenus unicolor japonicus Reitter. Several khapra beetle-specific mtDNA regions were identified and selected for use in the real-time PCR identification of these beetles based on Taq-Man® chemistry. Specificity tests conducted with the DNAs of several insects, including those of the three beetles described above, revealed that the primer/probe sets designed for the real-time PCR were highly specific to their targets.

A single fluorophore ratiometric nanosensor based on dual-emission DNA-templated silver nanoclusters for ultrasensitive and selective Pb2+ detection

Lead ion (Pb2+) is known as a major non-degradable environmental pollutant and is quite harmful to human health. In our investigation, a novel ratiometric fluorescent nanosensor with DNA-AgNCs as single fluorophore has been successfully constructed for ultrasensitive and specific detection of Pb2+. The single-strand DNA templated silver nanoclusters present green emission, which can be converted into red emission after approaching a specific DNA segment by forming duplex with its complementary DNA strands (ds-DNA-AgNCs). The ds-DNA-AgNCs contains rA cleavage site of Pb2+-dependent DNAzyme configuration. In the presence of Pb2+, the specific DNA segment would be released from the ds-DNA-AgNCs, resulting in discoloration of DNA-AgNCs from red to green. Since Pb2+ can successively cleave the rA site, the signal change from red to green emission is amplified. Thus, benefiting from the Pb2+-dependent DNAzyme and the charming properties of DNA-AgNCs, the proposed platform exhibits a good linear relationship from 0.001 nM to 10 nM with a detection limit of 1.0 pM for Pb2+ determination, which is lower than most of the reported Pb2+ biosensors. Moreover, this sensitive ratiometric fluorescent probe exhibits excellent selectivity toward Pb2+ detection in real samples, such as lake water, tap water, and human serum samples, illustrating the huge potential applications for complicated samples in the future.

Adaptive selection of founder segments and epistatic control of plant height in the MAGIC winter wheat population WM-800

BackgroundMulti-parent advanced generation intercross (MAGIC) populations are a newly established tool to dissect quantitative traits. We developed the high resolution MAGIC wheat population WM-800, consisting of 910 F4:6 lines derived from intercrossing eight recently released European winter wheat cultivars.ResultsGenotyping WM-800 with 7849 SNPs revealed a low mean genetic similarity of 59.7% between MAGIC lines. WM-800 harbours distinct genomic regions exposed to segregation distortion. These are mainly located on chromosomes 2 to 6 of the wheat B genome where founder specific DNA segments were positively or negatively selected. This suggests adaptive selection of individual founder alleles during population development. The application of a genome-wide association study identified 14 quantitative trait loci (QTL) controlling plant height in WM-800, including the known semi-dwarf genes Rht-B1 and Rht-D1 and a potentially novel QTL on chromosome 5A. Additionally, epistatic effects controlled plant height. For example, two loci on chromosomes 2B and 7B gave rise to an additive epistatic effect of 13.7 cm.ConclusionThe present study demonstrates that plant height in the MAGIC-WHEAT population WM-800 is mainly determined by large-effect QTL and di-genic epistatic interactions. As a proof of concept, our study confirms that WM-800 is a valuable tool to dissect the genetic architecture of important agronomic traits.

Monitoring of the Epidemic Situation with Q Fever in the Regions of Ukraine

ObjectiveTo investigate Q fever pathogen distribution among ixodic ticks, myomorphic rodents, febrile patients, residents of enzootic areas with Q fever and persons in contact with Q fever, specifically infected persons in the Southern and Western regions of Ukraine.IntroductionImprovement of the Q fever epizootic and epidemiological surveillance system remains an urgent veterinary service and healthcare problem in Ukraine. The grounds for this should be laid by the results of monitoring studies of persons with a professional infection risk (livestock farms, animal processing enterprises, veterinary specialists, etc.) and living in enzootic territories , as well as research of Q fever pathogen possible sources reservoirs.MethodsReal-time PCR - detection of specific DNA segments of Coxiella burnetii with application of commercial reagent kits. Immunofluorescence microscopy - detection of antigens/antibodies of studied rickettsia in biological substrates using luminescent immune sera labeled with fluorescein-5-isothiocyanate. Epidemiological methods - analysis of infectious diseases foci epidemiological maps. Statistical methods - data analysis using such software as Excel and Quantum GIS (1.6.0).ResultsPrimarily, Q fever endemic areas are formed because of the circulation of Coxiella burnetii in warm-blooded animal populations and their blood-sucking ectoparasites, which are the main source of the infection in humans. Based on the aggregated data received from multi-year research projects in Ukraine, Q fever enzootic territories were found in 18 administrative regions, Crimea and the city of Sevastopol. Currently we know of 257 areas where the pathogen was detected. The epidemic process in these territories is manifested by sporadic human diseases and the detection of the pathogen in natural carriers. The possibility of the natural foci epidemic potential increase in these territories is confirmed by the higher titers of Q fever pathogen specific antibodies detected in the local population.The results of the research of the infected material that was collected in Southern Ukraine during 2014-2016, showed the preservation of the Q fever causative agent in natural foci both in Danube-Dniester interfluve area of Odesa region and in Trans-Dnistrer areas, and its significantly less prevalent in the area adjacent to Odessa. In addition, the signs of natural foci formation have been revealed in other areas, which is indicative of current epidemic activity of natural foci of the infection. The results of serological studies and clinical and epidemiological surveys indicate that in the immunological structure of the population of the Danube-Dniester interfluve areas of Odessa region, Q fever is most common in rural population of working age, especially those constantly contact with farm animals. In the Ivano-Frankivsk region, serological studies in 2014 -2016, detected no Q fever seropositive people, indicating the pathogen being in the reserve stage, which corresponds to the inter-epidemic period. At the same time, the detection of C. burnetii in ticks in the enzootic territories indicates the possibility of the pre-epidemic process being formed.Since by pathogen range and transmission mechanisms Q fever in Ukraine is associated with many natural-focal zoonotic infections, it is advisable to monitor endemic areas using a modern observation algorithm using the introduction of geoinformation systems and the molecular genetic characteristics of circulating strains. This will increase the effectiveness of the detection of current natural and anthropurgic foci of such infections, will contribute to their detailed characterization and systematization, improve epidemiological surveillance and prevent the emergence of epidemic outbreaks among the population. The results of the research will contribute to the improvement of differential diagnosis of febrile states with an unclear etiologic agent.ConclusionsThe results of the Q fever pathogen detection in the material collected in Southern and Western regions of Ukraine showed that the area of prevalence of this agent has been expanded to the areas and settlements that are not included in the list of enzootic territories. Involvement in the ecological cycles of ixodic ticks and mouse-like rodents was observed. The presence of polyvectoral and polyhostal natural foci of this infection was found. The circulation of the causative agent of Q fever in the territories of Odesa and Ivano-Frankivsk regions where epidemic outbreaks and sporadic disease in people were also observed.

A DNA segment encoding the anticodon stem/loop of tRNA determines the specific recombination of integrative-conjugative elements in Acidithiobacillus species

ABSTRACTHorizontal gene transfer is crucial for the adaptation of microorganisms to environmental cues. The acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans encodes an integrative-conjugative genetic element (ICEAfe1) inserted in the gene encoding a tRNAAla. This genetic element is actively excised from the chromosome upon induction of DNA damage. A similar genetic element (ICEAcaTY.2) is also found in an equivalent position in the genome of Acidithiobacillus caldus. The local genomic context of both mobile genetic elements is highly syntenous and the cognate integrases are well conserved. By means of site directed mutagenesis, target site deletions and in vivo integrations assays in the heterologous model Escherichia coli, we assessed the target sequence requirements for site-specific recombination to be catalyzed by these integrases. We determined that each enzyme recognizes a specific small DNA segment encoding the anticodon stem/loop of the tRNA as target site and that specific positions in these regions are well conserved in the target attB sites of orthologous integrases. Also, we demonstrate that the local genetic context of the target sequence is not relevant for the integration to take place. These findings shed new light on the mechanism of site-specific integration of integrative-conjugative elements in members of Acidithiobacillus genus.

CRISPR-mediated isolation of specific megabase segments of genomic DNA.

Megabase-sized, complex, repetitive regions of genomes are poorly studied, due to the technical and computational challenges inherent to both assembling precise reference sequences and accurately assessing structural variation across contiguous megabase DNA regions. Here we describe a strategy to overcome these challenges, CISMR (CRISPR-mediated isolation of specific megabase-sized regions of the genome), which enables us to perform targeted isolation of contiguous megabase-sized segments of the genome. Direct sequencing of the purified DNA segments can have >100-fold enrichment of the target region, thus enabling the exploration of both DNA sequence and structural diversity of complex genomic regions in any species.

CRISPR edits human embryos

For the first time in the U.S., researchers have used the CRISPR/Cas9 gene-editing system in human embryos to correct a harmful hereditary gene mutation. Led by Shoukhrat Mitalipov of Oregon Health & Science University, the team injected eggs from healthy donors with sperm from a donor with hypertrophic cardiomyopathy, a common condition characterized by irregular heartbeat and heart failure. People with the disease carry a mutation in one of two copies of their MYBPC3 gene. The researchers simultaneously injected CRISPR/Cas9 components with the sperm to repair the mutation and produce embryos with two healthy copies of the gene. CRISPR/Cas9 uses guide-RNA molecules to target and cut specific segments of DNA. Natural DNA-repair mechanisms in the cell follow up by filling in the missing pieces. Mitalipov and colleagues found that in embryos coinjected with sperm and CRISPR components, a highly accurate DNA repair mechanism used the eggs’ healthy copy of the

Structural basis for DNA recognition by STAT6

STAT6 participates in classical IL-4/IL-13 signaling and stimulator of interferon genes-mediated antiviral innate immune responses. Aberrations in STAT6-mediated signaling are linked to development of asthma and diseases of the immune system. In addition, STAT6 remains constitutively active in multiple types of cancer. Therefore, targeting STAT6 is an attractive proposition for treating related diseases. Although a lot is known about the role of STAT6 in transcriptional regulation, molecular details on how STAT6 recognizes and binds specific segments of DNA to exert its function are not clearly understood. Here, we report the crystal structures of a homodimer of phosphorylated STAT6 core fragment (STAT6CF) alone and bound with the N3 and N4 DNA binding site. Analysis of the structures reveals that STAT6 undergoes a dramatic conformational change on DNA binding, which was further validated by performing molecular dynamics simulation studies and small angle X-ray scattering analysis. Our data show that a larger angle at the intersection where the two protomers of STAT meet and the presence of a unique residue, H415, in the DNA-binding domain play important roles in discrimination of the N4 site DNA from the N3 site by STAT6. H415N mutation of STAT6CF decreased affinity of the protein for the N4 site DNA, but increased its affinity for N3 site DNA, both in vitro and in vivo. Results of our structure-function studies on STAT6 shed light on mechanism of DNA recognition by STATs in general and explain the reasons underlying STAT6's preference for N4 site DNA over N3.

A disposable, continuous-flow polymerase chain reaction device: design, fabrication and evaluation

Polymerase Chain Reaction (PCR) is used to amplify a specific segment of DNA through a thermal cycling protocol. The PCR industry is shifting its focus away from macro-scale systems and towards micro-scale devices because: micro-scale sample sizes require less blood from patients, total reaction times are on the order of minutes opposed to hours, and there are cost advantages as many microfluidic devices are manufactured from inexpensive polymers. Some of the fastest PCR devices use continuous flow, but they have all been built of silicon or glass to allow sufficient heat transfer. This article presents a disposable polycarbonate (PC) device that is capable of achieving real-time, continuous flow PCR in a completely disposable polymer device in less than 13 minutes by thermally cycling the sample through an established temperature gradient in a serpentine channel. The desired temperature gradient was determined through simulations and validated by experiments which showed that PCR was achieved. Practical demonstration included amplification of foot-and-mouth disease virus (FMDV) derived cDNA.