Produced DNA Fragments Research Articles

Allergen-stimulated secretion of DNA fragments by the airway epithelium enhances type 2 immune responses

Exposure of human bronchial epithelial cells (hBECs) and mouse airways to allergens from the fungus Alternaria alternata triggers the release of alarmin signaling molecules including ATP, IL-33 and DNA fragments. The goal of this study was to determine the mechanisms that regulate release of genomic DNA fragments and how extracellular DNA (eDNA) contributes to type 2 immunity. Treatment of hBECs with Alternaria extract induces oxidative stress, which leads to ATP release and uptake of Ca 2+ following activation of P2X receptors. DNA release was dependent on a sustained increase in intracellular [Ca 2+ ], which stimulates cleavage of caspase 3 by the proprotein convertase enzyme furin, resulting in activation of caspase 3 and ultimately nuclear DNA fragmentation as shown in comet assays. Some of the fragmented DNA was released into the media under conditions where barrier function of the epithelium was maintained, and nuclear and plasma membranes remained intact. Alternaria-induced DNA release was detected within 30 minutes following exposure, reached its peak at 4 hours and ceased after 12 hours. Pretreatment of hBECs with a calpain inhibitor resulted in continued DNA release and loss of cell viability by 24 hours. This result suggested that calpain activity contributed to termination of Alternaria-evoked DNA release. In mice, intranasal administration of Alternaria extract produced release of DNA fragments into bronchoalveolar lavage (BAL) fluid over a similar time course as observed in hBECs. In caspase 3 deficient mice however, DNA release into BAL fluid was significantly reduced. Furthermore, Alternaria-induced type 2 immune responses were suppressed following pretreatment with a DNA scavenger. Conversely, intranasal administration of mouse genomic DNA with Alternaria amplified IL-5 and IL-13 secretion into BAL fluid while DNA alone had no effect. These findings indicate that DNA release from the airway epithelium is a regulated process involving noncanonical activation of caspase 3 to produce DNA fragmentation and calpain activity, which appears to be essential for the termination of DNA release. Moreover in airways, eDNA appears to function as a secondary alarmin that acts on immune cells to enhance type 2 immunity. This work was supported by grants from the NIH (AI128729 and AI169530) to HK and SMO. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High-molecular-weight DNA extraction for long-read sequencing of plant genomes: An optimization of standard methods.

Developing an effective and easy-to-use high-molecular-weight (HMW) DNA extraction method is essential for genomic research, especially in the era of third-generation sequencing. To efficiently use technologies capable of generating long-read sequences, it is important to maximize both the length and purity of the extracted DNA; however, this is frequently difficult to achieve with plant samples. We present a HMW DNA extraction method that combines (1) a nuclei extraction method followed by (2) a traditional cetyltrimethylammonium bromide (CTAB) DNA extraction method for plants with optimized extraction conditions that influence HMW DNA recovery. Our protocol produced DNA fragments (percentage of fragments >20 kbp) that were, on average, ca. five times longer than those obtained using a commercial kit, and contaminants were removed more effectively. This effective HMW DNA extraction protocol can be used as a standard protocol for a diverse array of taxa, which will enhance plant genomic research.

The Use of Primers Heavily Labeled with Fluorescein in Polymerase Chain Reaction

The fluorescently-labeled DNA is widely used in various bioanalytical applications. For a number of applications, a high level of labeling could be beneficial. One of the ways to produce DNA fragments bearing multiple fluorescent tags is to use polymerase chain reaction (PCR) with primers heavily labeled with fluorophores. Here we tested how primers with multiple fluorescein tags perform in PCR. It has been found that the positioning of fluorescein tags at or near the 3'-end upon primer multiple labeling can inhibit DNA amplification (up to a complete stop when tags are placed at the 3'- or adjacent nucleotide). The mechanism, by which the presence of fluorescein tags at or near the primer 3'-end affects the PCR performance, is rather ambiguous and can involve both a steric hindrance for polymerase binding from the fluorescein moiety, as well as destabilization of a primer-template duplex. Nonetheless, if multiple fluorescein tags are attached so that at least three nucleotides from the primer 3'-end are unmodified, the production of DNA fragments bearing multiple fluorescein molecules is possible even if both primers are heavily labeled, though on the expense of amplicon yield.

In Vitro Evaluation of Anti-Rotaviral Activity and Intestinal Toxicity of a Phytotherapeutic Prototype of Achyrocline bogotensis (Kunth) DC.

Viruses represent the primary etiologic agents (70-80%) of acute diarrheal disease (ADD), and rotavirus (RV) is the most relevant one. Currently, four rotavirus vaccines are available. However, these vaccines do not protect against emerging viral strains or are not available in low-income countries. To date, there are no approved drugs available against rotavirus infection. In this study, we evaluated the in vitro anti-rotaviral activity and intestinal toxicity of a phytotherapeutic prototype obtained from Achyrocline bogotensis (Kunth) DC. (PPAb); medicinal plant that contains compounds that inhibit the rotavirus replication cycle. Virucidal and viral yield reduction effects exerted by the PPAb were evaluated by immunocytochemistry and flow cytometry. Furthermore, the toxic impact of the PPAb was evaluated in polarized human intestinal epithelial C2BBe1 cells in terms of cytotoxicity, loss of cytoplasmic membrane asymmetry, and DNA fragmentation by MTT and fluorometry. PPAb concentrations under 0.49 mg/mL exerted significant virucidal and viral yield reduction activities, and concentrations under 16 mg/mL neither reduced cell viability, produced DNA fragmentation, nor compromised the C2BBe1cell membrane stability after 24-h incubation. Based on these results, the evaluated phytotherapeutic prototype of Achyrocline bogotensis might be considered as a promising alternative to treat ADD caused by rotavirus.

Cadmium-induced ultrastructural changes and apoptosis in the gill of freshwater mussel Anodonta woodiana.

This study investigated the acute toxicity of cadmium (Cd) to the freshwater mussel Anodonta woodiana. The freshwater mussels were exposed to five concentrations of Cd (0mg/L, 8.43mg/L, 16.86mg/L, 33.72mg/L, and 67.45mg/L) for up to 96h. The 24-h, 48-h, 72-h, and 96-h LC50 values for Cd were estimated as 562.3mg/L, 331.1mg/L, 182.0mg/L, and 134.9mg/L, respectively. Caspase-3, caspase-8, caspase-9, and Ca-ATPase activities; protein and H2O2 levels; DNA fragmentation; and ultrastructure of the gill were also investigated. The activities of caspase-3 and caspase-9 in mussels were increased by Cd in a dose-dependent manner, where higher doses of Cd (33.72mg/L and 67.45mg/L) significantly increased the enzyme activities compared to the controls (P < 0.05). The caspase-8 activity was significantly depressed by a low dose of Cd (8.43mg/L) but was clearly induced by higher doses of Cd (16.86mg/L, 33.72mg/L, and 67.45mg/L) (P < 0.05). The Ca-ATPase activity and H2O2 levels were elevated and reached maximum values under the medium dose of Cd (16.86mg/L). However, protein levels were decreased by Cd in an inverse dose-dependent manner. In the gills of the mussels, Cd treatment induced DNA fragmentation as demonstrated by DNA ladders observed via agarose gel electrophoresis. Moreover, ultrastructural alterations in gill cells of mussels treated with Cd (16.86mg/L and 67.45mg/L) for 96h were observed by electronic microscopy. The ultrastructure abnormalities were characterized by the following features: (1) a disordered arrangement and breaking off of microvilli of epithelial cells; (2) chromatin condensed near the nuclear membrane and the appearances of extremely irregular nuclei, some with a fingerlike shape and an unclear, swollen, invaginated, or ruptured nuclear membrane and apoptotic bodies; (3) swollen and vacuolating mitochondria, some with disintegrated or missing cristae; (4) a disintegrated rough endoplasmic reticulum containing different sizes of vesicles; and (5) shrinking and deformation of Golgi bodies with decreased vesicle numbers. Our results demonstrated that Cd could activate caspase-3, caspase-8, caspase-9, and Ca-ATPase; cause ultrastructural changes; and produce DNA fragmentation in the mussels investigated. Based on the information obtained through this study, it is reasonable to conclude that Cd can induce apoptosis in the gills of the mussels, eventually leading to tissue damage.

Chemical Composition, α-Glucosidase Inhibitory and Anticancer Activity of Essential Oil of Thymus vulgaris Leaves

Dietary herbs are used for imparting flavour, aroma, and piquancy to foods. Besides their role in improving food quality, spices also have health benefits, such as anticancer, antidiabetic, antimicrobial, antioxidant, hypolipidemic, analgesic, immunostimulant, etc. Thymus vulgaris (family: Lamiaceae) is a valuable medicinal herb known for its health benefits. The essential oil from T. vulgaris was isolated by the hydrodistillation method, and chemical constituents in it were determined by GC-MS. Anticancer activity was determined using a DNA ladder assay, and antidiabetic activity was accessed by α-glucosidase inhibition. The GC-MS analysis showed the presence of 16 chemical constituents, and thymol (55.88 %), linalool (13.71 %), carvacrol (8.36 %), and p-cymene (6.00 %) were found as major components. The antidiabetic activity of T. vulgaris essential oil showed potent inhibition of α-glucosidase enzyme with an IC50 value 125.1±4.25 μg/mL. The anticancer activity results revealed that treating MCF-7 cells with 100 μg/mL essential oil of thyme produces DNA fragmentation and promotes apoptosis. The current study provides a scientific basis for its possible use in controlling diabetes and breast cancer. The study further expands the knowledge of T. vulgaris chemical composition and provides evidence of ethnomedicinal use of the plant by exploring antidiabetic and anticancer activities.

The universal dual-mode aptasensor for simultaneous determination of different bacteria based on naked eyes and microfluidic-chip together with magnetic DNA encoded probes

It was critically important to develop some sensitive, convenient and on-site methods for simultaneous assay of different pathogenic bacteria in foods. In this work, a dual-mode aptasensor was established for fulfilling above aims combing colorimetry with microfluidic chip. This as-prepared dual-mode aptasensor not only realized rapid screening by naked eye on-site, but also the simultaneous quantification of multiple bacteria. Namely, the presence of pathogenic bacteria was firstly judged by naked eyes with Salmonella typhimurium (S.T) and Vibrio parahaemolyticus (V.P) as models. And then, S.T and V.P in positive samples were simultaneously quantified by microfluidic chip. In order to obtain the multiple signals, a series of magnetic DNA encoded-probes (MDEs) was fabricated containing rolling cycle amplified long DNA chain (RCA-DNA) rich in G-quadruplex sequences. They can combine with hemin as DNAzyme to catalyze 3,3′-5,5′-Tetramethyl benzidine (TMB)-H2O2 system for color development and be cleaved by EcoRV endonuclease to produce DNA fragments with different lengths. The microfluidic chip was employed to separate and quantify the fragments for quantifying S.T and V.P simultaneously. For this protocol, 100 CFU·mL−1 of V.P or S.T could be observed by the naked eye and as low as 32 S.T and 30 CFU·mL−1 V.P could be detected by the chip within 3 min. The dual-mode aptasensor could quickly screen positive samples, and simultaneously perform quantitative detection of the bacteria in positive samples. Our protocol demonstrated its potential in on-site qualification & simultaneous quantification of foodborne bacteria in foods.

Identification and diversity analysis of cassiicolin-encoding gene of Corynespora cassiicola isolates from rubber tree in Indonesia

Abstract. Oktavia F. 2020. Identification and diversity analysis of cassiicolin encoding gene of Corynespora cassiicola isolates from rubber tree in Indonesia. Biodiversitas 21: 3499-3507. Cassiicolin is an important effector of virulence process on the Corynespora cassiicola, plant-pathogen that causes Corynespora leaf fall disease in rubber tree. The study aimed to identify and analyze the genetic diversity of isolates from rubber plants in Indonesian based on cassiicolin encoding gene (Cas gene). Identification of Cas genes was performed using 12 pairs of primers on 23 isolates from rubber host clones. Only three pairs of primers were able to amplify the analyzed DNA, namely CasF20/CasR28, CasF19/CasR26, and CasF4/R16 which produced DNA fragments of 760 bp, 550 bp and 210 bp respectively in this study. BLAST analysis showed that 19 isolates carried a Cas5 gene. Three of the 19 DNA samples also contained a putative Cas3 or Cas4 gene. Four other isolates had no detectable Cas gene (Cas0). The genetic diversity based on the Cas gene showed that the isolates were grouped into three major clusters. ome isolates had high genetic similarity with outgroup isolates from China. There was not clear link between Cas gene with virulence profiles. The cassiicolin encoding gene information is useful for predicting the virulence of C. cassiicola isolates from Indonesia rubber plantation, so that the disease severity spreading in rubber plants can be early anticipated.

Isolation and Amplification DNA on Endophytic Fungi of Local Aromatic Rice Enrekang

Local rice is one of the biodiversity that is currently not explored in detail. The exploration of endophytic fungi of local rice in South Sulawesi was carried out to find out the types of endophytic fungi in the local rice plant. Plant samples were taken from local rice plantations in the Tator, Enrekang, and Luwu regencies. DNA isolation and PCR amplification were the initial stages that need to be carried out in molecular identification. DNA fungi extraction was performed by using DNesay DNA extraction KIT. The result of DNA fungi isolation was electrophoresed with 0.8% agarose gel in TAE buffer (Tris Acetate EDTA). DNA isolation was successfully carried out by the formation of DNA bands. DNA amplification of two endophytic fungi using the primary pairs of ITS1 and ITS4, with condition PCR of predenaturation at 95°C for 5 minute, denaturation at 94°C for 1 minute, annealing at 55,4°C for 1 minute, elongation at 72°C for 1 minute, final elongation at 72°C for 10 minute. The denaturation stage until the extension was repeated 35 times and produced DNA fragments with a molecular size of 700 bp and 600 bp.

Complex between a Multicrossover DNA Nanostructure, PX-DNA, and T7 Endonuclease I.

Paranemic crossover DNA (PX-DNA) is a four-stranded multicrossover structure that has been implicated in recombination-independent recognition of homology. Although existing evidence has suggested that PX is the DNA motif in homologous pairing (HP), this conclusion remains ambiguous. Further investigation is needed but will require development of new tools. Here, we report characterization of the complex between PX-DNA and T7 endonuclease I (T7endoI), a junction-resolving protein that could serve as the prototype of an anti-PX ligand (a critical prerequisite for the future development of such tools). Specifically, nuclease-inactive T7endoI was produced and its ability to bind to PX-DNA was analyzed using a gel retardation assay. The molar ratio of PX to T7endoI was determined using gel electrophoresis and confirmed by the Hill equation. Hydroxyl radical footprinting of T7endoI on PX-DNA is used to verify the positive interaction between PX and T7endoI and to provide insight into the binding region. Cleavage of PX-DNA by wild-type T7endoI produces DNA fragments, which were used to identify the interacting sites on PX for T7endoI and led to a computational model of their interaction. Altogether, this study has identified a stable complex of PX-DNA and T7endoI and lays the foundation for engineering an anti-PX ligand, which can potentially assist in the study of molecular mechanisms for HP at an advanced level.

A simple method for distinguishing Bursaphelenchus xylophilus, B. mucronatus mucronatus, and B. m. kolymensis (Nematoda: Aphelenchoididae) by polymerase chain reaction with specific primers designed based on cytochrome oxidase subunit I genes

Based on DNA sequences of mitochondrial cytochrome oxidase subunit I (mtCOI) genes, three PCR forward primers specific for Bursaphelenchus mucronatus mucronatus, B. m. kolymensis, and B. xylophilus (Nematoda: Aphelenchoididae), and one common PCR reverse primer were developed to determine the interspecific hybrids in a population. PCR amplification with the new primer pairs was expected to produce DNA fragments of 286 bp, 117 bp and 479 bp for B. m. mucronatus, B. m. kolymensis, and B. xylophilus, respectively. Electrophoresis of PCR products with the new primer pairs distinguished the two B. mucronatus subspecies and B. xylophilus collected in Japan. It also exhibited a band-free image for Bursaphelenchus doui, B. niphades, B. sinensis, B. firmae, Bursaphelenchus sp. (NK230), and Aphelenchoides stammeri, whereas it exhibited one (ca. 100-bp DNA fragment) and two (ca. 100 and 500-bp DNA fragments) bands for B. poligraphi and B. yongensis, respectively. As PCR products with other primer pairs for nuclear ribosomal RNA (rRNA) gene distinguished B. mucronatus (210 bp DNA fragment), B. xylophilus (557 bp fragment), and the eight other nematode species (no amplification), primer pairs for mtCOI and rRNA genes can determine whether B. poligraphi resides in specified trees or in study pine stands. Thus, those primer pairs would be helpful to find out hybrids between B. mucronatus and B. xylophilus and to analyze the dynamics of hybrids and introgressants in hybrid-derived populations in field and laboratory.

PCR Multipleks untuk Identifikasi Mycobacterium tuberculosis Resisten terhadap Isoniazid dan Rifampisin pada Galur Lokal Balai Laboratorium Kesehatan Provinsi Jawa Barat

The incidence of multidrug-resistant tuberculosis (MDR-TB) cases has become the biggest source of the problem in the effort to eradicate tuberculosis (TB) disease in Indonesia. MDR-TB is a resistant TB bacteria to the two, at least, first-line TB drugs, e.g., rifampin and isoniazid. Unfortunately, the current diagnostics methods to identify the MDR-TB are still slow, unspecific, and inaccurate. The purpose of this study is to identify the isoniazid- and rifampin-resistant M. tuberculosis (local strain Balai Laboratorium Kesehatan Provinsi Jawa Barat) by using multiplex PCR method. The TB bacteria colonies were cultivated in Middlebrook 7h9 broth media, which followed by the isolation of chromosomal DNA. The best PCR condition was achieved by optimizing the annealing temperature, the concentration of magnesium chloride, and a number of the cycle. Multiplex PCR was conducted with inhA1-inhA2, rpoB1- rpoB2, katG1- katG2, and B1-B2 pair primers. Furthermore, the PCR product was characterized on 2% gel agarose electrophoresis which stained by using ethidium bromide. The result showed that isoniazid- and rifampin-resistant M. tuberculosis sample could be identified using multiplex PCR, producing DNA fragments with a size of 71 bp, 124 bp 186 bp, and 200 bp. A non-MDR-TB only produced one DNA fragments with a size of 200 bp. Therefore, it can be concluded that MDR-TB and non-MDR-TB can be distinguished using multiplex PCR with a combination of four pair primers.

Far rapid synthesis of giant DNA in the Bacillus subtilis genome by a conjugation transfer system

Bacillus subtilis offers a platform for giant DNA synthesis, which is mediated by the connection of overlapping DNA segments called domino DNA, in the cloning locus of the host. The domino method was successfully used to produce DNA fragments as large as 3500 kbp. However, domino DNA is limited to <100 kbp because of size restrictions regarding the transformation (TF) of B. subtilis competent cells. A novel conjugal transfer (CT) method was designed to eliminate the TF size limit. The CT method enables rapid and efficient domino reactions in addition to the transfer of giant DNA molecules of up to 875 kbp to another B. subtilis genome within 4 hours. The combined use of the TF and CT should enable significantly rapid giant DNA production.

Use of a modified AFLP protocol to discriminate Salmonella enterica subsp.enterica serovar Enteritidis isolates

Salmonella enterica subsp. enterica ( S .) serovar Enteritidis is one of the main pathogens involved in food-borne diseases worldwide. In epidemiological investigations of food-related salmonellosis, subtyping is necessary to improve preventive and control measures. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis is a modified AFLP that uses only one restriction enzyme to produce DNA fragments that are selectively amplified by PCR. In order to assess the applicability of SE-AFLP in S. Enteritidis typing, one hundred and eight strains isolated from poultry, swine and also from human salmonellosis outbreaks in Southern Brazil were analyzed. Strains from other countries and six different S. enterica serovars were also included as controls. SE-AFLP was able to distinguish S . Enteritidis from the other S. enterica serovars analyzed. However, most of S . Enteritidis strains isolated from poultry, salmonellosis outbreaks and most of the strains from other countries shared the same predominant pattern. The low genetic diversity identified in S . Enteritidis suggests that the strains analyzed are clonally related and one predominant SE-AFLP genotype is widely spread in Southern Brazil.

Assessment of resistance status of some tomato genotypes to bacterial wilt disease and evaluation of SNP marker (LEOH19) for selection of BW resistant gene

Marker assisted selection (MAS) has become very important and useful in selection of disease resistance genes in crop plants. Tomato, Solanum lycopersicum, is one of the most important vegetables worldwide but its production is being affected by pests and diseases, one of which is bacterial wilt caused by Ralstonia solanacearum. In endemic areas, the disease constitutes up to 100% yield loss. A-Two-season completely randomized design (CRD) experimental trial was conducted to assess the resistance status of forty (40) tomato genotypes. The results showed variations in resistance status, from highly resistance to highly susceptible. DNA concentrations from the assessed genotypes ranged between 14.46 and1430.52 ng/ul. Single Nucleotide Polymorphism (SNP) marker, LEOH19, was evaluated on the forty tomato genotypes for the identification of bw-gene that confers resistance to R. solanacearum. Genomic DNA was amplified using the primer sequence; (forward primer; 5´- AAGGCTCAGAAAGGGTCCAT-3´, reverse primer; 5´- GAGTTCATCAACACATCACACA-3´). The primer pairs produced amplification at 300 bp in 35 genotypes. After digestion, the product produced 300 bp in 33 genotypes. In screenhouse study, AVTO9803, AVTO0201, Tomachiva and Eyetom were found highly resistant and could be considered good materials in grafting and breeding programme for bacterial wilt resistance development. The results indicated that the primer amplified the specific sequence of the bw-gene locus in both the resistant and susceptible alleles. LEOH19 was found to be monomorphic and produced DNA fragments in both resistant and susceptible tomato genotypes and could, therefore, not be used as a molecular marker for marker assisted selection in tomato breeding programme. Key words : Bacterial wilt, Ralstonia solanacearum, resistance, SNP, Tomato.

Designing and Reconstruction of pcDNA 3.0 Mammalian Expression Vector with its Multiple Cloning Sites by Directional Cloning Method

Traditional cloning is the cloning in which we use restriction endonucleases to produce DNA fragments with specific complementary end sequences that can be joined together with a DNA ligase enzyme, prior to the transformation. The purpose of this study was to reconstruct the mammalian expression vector by replacing the junk DNA with its natural multiple cloning sites. Plasmid was propagated and digested with Xho1 and Kpn1 enzymes from both sides of junk DNA a 600 bp DNA was cut from the vector and then ligation of 30 bp primer which was designed similar as its multiple sequence sites. Correct insertion was confirmed by plasmid isolation, plasmid colony PCR, PCR of cloned plasmid and comparison on gel electrophoresis with the original one and finally by sequencing. An extra DNA fragment of 600 bp was cut from the vector after restriction digestion. Ligated colonies appeared on the agar plate from which 60 bp colony PCR products was produced. Plasmid isolation after cloning and colony PCR and PCR of cloned plasmid confirmed the cloning. Cloning of multiple cloning sites in pcDNA3.0 mammalian expression vector was performed successfully. Ligation of multiple cloning sites in pcDNA vector provided various restriction enzymes recognition sites for simple and fast cloning.

Food Analysis Using Organelle DNA and the Effects of Processing on Assays.

Extrachromosomal DNA such as organelle DNA are increasingly targeted in molecular detection assays where samples have been degraded by physical or chemical means. Owing to multiple organelles per cell and greater copy numbers than nuclear genes, organelle gene targets provide a more robust signal in polymerase chain reaction (PCR), quantitative PCR (qPCR), and other emerging molecular technologies. Because of these advantages, direct analysis of organelle DNA in food matrices is used for detection of contaminants and identification and authentication of food ingredients and allergens. Non-nuclear DNA is also used as an assay normalizer for detection of genetically modified organisms (GMOs) in foods. This review describes these protocols plus the effects of processing on efficacy, with special emphasis on thermally produced DNA fragmentation. Future research may incorporate molecular techniques beyond detection, used instead as time-temperature indicators in thermal food processing or quality indicators in food fermentation or acidification.

The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity.

BackgroundThe ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly.ResultsRSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3’-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %.ConclusionsRSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing templates amenable to DNA sequencing on recently developed platforms. Although our demonstration of the method does not utilize these DNA sequencing platforms directly, our results indicate that the capture of long DNA fragments produce superior coverage of the targeted region.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2836-6) contains supplementary material, which is available to authorized users.

Role of constitutive calcium-independent phospholipase A2 beta in hippocampo-prefrontal cortical long term potentiation and spatial working memory

Calcium independent phospholipase A2 (iPLA2) is an 85kDa protein that catalyzes the hydrolysis of the sn-2 acyl ester bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. In this study, we determined the role of constitutive iPLA2β in long term potentiation (LTP) of the hippocampo-prefrontal cortical pathway in vivo. We also examined the effect of iPLA2β knockdown using the rewarded alternation in T-maze task, a test of spatial working memory which is dependent on this pathway. Intracortical injection of an inhibitor to iPLA2, bromoenol lactone (BEL) or antisense oligonucleotide to iPLA2β in the prefrontal cortex abolished induction of hippocampo-prefrontal cortical LTP. Moreover, iPLA2 inhibition and antisense knockdown resulted in increased errors in the rewarded alternation in T-maze task, indicating negative effects on spatial working memory. BEL or antisense injection did not produce DNA fragmentation in the cortex as demonstrated by TUNEL assay. Results confirm a role of constitutive iPLA2β in hippocampo-prefrontal cortical synaptic plasticity in vivo, and add to previous observations of a role of iPLA2 in hippocampal LTP in vitro, and long-term memory retrieval. They may be relevant in Alzheimer’s disease, and other neurodegenerative conditions that are associated with changes in iPLA2.

Application of Pulsed Field Gel Electrophoresis for Study of Genetic Diversity in Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients.

Background:Mycobacterium tuberculosis genotyping can effectively improve tuberculosis (TB) control programs by controlling disease transmission. Pulsed field gel electrophoresis (PFGE) is a particularly powerful tool for determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease.Objectives:The aim of present study was to investigate the genetic diversity of M. tuberculosis strains in Khuzestan province by the PFGE technique.Patients and Methods:In total, 80 M. tuberculosis positive cultures were obtained from tuberculosis patients. PFGE was performed on 60 PCR-confirmed isolates by using DraI and XbaI restriction enzymes according to standard protocols. Plugs containing digested DNA were then loaded on agarose gels and run using contour-clamped homogenous electric fields.Results:Fifty distinct DNA banding patterns were obtained by digestion of DNA with DraI and 38 DNA banding patterns by digestion with XbaI restriction enzymes. The patterns comprised of 17 different clusters in which cluster I was the major one, containing six strains. Three clusters contained three strains each and the 13 remaining clusters comprised of two strains each. Digestion with DraI yielded 15-20 DNA fragments with 50-485 kb size, while digestion by XbaI produced DNA fragments with a size smaller than 50-242 kb.Conclusions:Despite the ability of PFGE for study of genetic diversity of many mycobacterial species and it being considered as a robust and useful tool, in this study we only found a 15% epidemiological relationship amongst the isolates. Thus, for higher discrimination of genotypic clusters among M. tuberculosis clinical isolates, the application of more sophisticated complementary techniques is required.