NEB Scientific Posters
Your search returned 82 results.
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Draft genome sequences of Mansonella perstans and Mansonella ozzardi and their Wolbachia endosymbionts (2019)
Mansoneliasis is a widespread, yet neglected, filariasis of humans caused by infection with Mansonella perstans, M. ozzardi and M. streptocerca. Transmission to humans is via midge and black fly insect vectors whose endosymbiont is a member of a unique Wolbachia supergroup that is from both insect and filarial hosts. In this study, draft genome sequences of M. perstans, M. ozzardi and Wolbachia were obtained. This will provide insight into the biology and evolution of some of the most neglected filarial parasites.
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Choosing the Right Exonuclease (2019)
Download our poster to find the exonuclease best suited for your application.
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EM-seq™ Enables Accurate and Precise Methylome Analysis of Challenging DNA Samples (2019)
Cell-free DNA (cfDNA) is gaining popularity as a noninvasive biomarker of disease. Most often, cfDNA is recovered from exosomes and other microvesicles released into body fluids (e.g., blood, urine, tears, etc.), providing an indicator of an organism’s health and/or disease. As methylation status has been shown to influence the progression of certain diseases, including cancer, analyzing the methylome of circulating cfDNA is an essential step.
Historically, the method of choice for methylome analysis was bisulfite sequencing, a method that leaves significant DNA damage in its wake. The NEBNext® Enzymatic Methyl-seq Kit (EM-seq™) enables higher quality library generation and improved sequence coverage, without added GC bias. To learn more about EM-seq after reviewing this poster, check out the tech note on this topic. -
A Single-tube, Low Input Protocol for Long Read Sequencing (2019)
Long read sequencing has become more popular with the advent of new technologies that support it. Both the PacBio® Sequel and Oxford Nanopore MinION™ offer platforms for long read sequencing, enabling simpler genome assembly, sequencing through complex regions, and identifying structural variants.
To fully exploit long read sequencing, researchers require a robust and reliable option for generating full-length cDNA from the source mRNA. The NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module is well-suited for upstream cDNA generation prior to a long read sequencing method. For additional details, please visit the product page. -
Uncovering the Cannabis sativa Methylome Through Enzymatic Methyl-seq (2019)
The medical and agricultural value of Cannabis is undeniable, and research is just now beginning to fully interrogate the plant and its products. Due to the base composition of the Cannabis genome (66% AT-rich), obtaining high-quality methylome analysis with whole genome bisulfite analysis (WGBS) has been challenging. WGBS is known to cause DNA damage that skews post-WGBS base composition (83% AT-rich).
NEBNext® Enzymatic Methyl-seq (EM-seq®) is an alternative to WGBS that does not require harsh conditions to accurately generate high-quality libraries, without losing sample integrity or details about methyl marks (both 5mC and 5hmC). With EM-seq, it becomes possible to analyze Cannabis plant tissues with greater accuracy and with less risk for DNA damage. For additional details on this study, please download this poster. -
Discovering the Maltose Binding Protein epitope tag for Escherichia coli expressed proteins (2018)
Maltose Binding Protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The anti-MBP monoclonal antibody B48 binds tightly and has no crossreactivity to other proteins in an E. coli lysate. For all these criteria, the MBP tag provides a useful epitope for fusion proteins expressed in E. coli.
The co-crystal structure of MBP bound to its antibody was solved and four amino acids of MBP were found to define the binding interaction. This epitope is the turn of an alpha helix packed by two beta strands. The failure to find a linear epitope by phage display suggests the helix-turnsheet is important in defining the smallest MBP epitope. Fusion of various fragments of MBP to the glutathione S-transferase protein was engineered in order to identify the smallest fragment, still recognized by the anti-MBP antibody. Further engineering of the epitope to stabilize and minimize the tag is in progress. -
Complete circular genome sequence of the Wolbachia wAlbB endosymbiont of Aedes albopictus (2018)
Wolbachia are α-proteobacteria belonging to the order Rickettsiales. It is a maternally transmitted, intracellular symbiont of arthropods and nematodes and estimated to infect 40-60% of arthropod species. The tiger mosquito Aedes albopictus is naturally infected with Wolbachia strains wAlbA and wAlbB. Cell line Aa23 established from Aedes albopictus embryos retains only wAlbB and is used as a key model to study host-endosymbiont interactions. The available wAlbB genome with 156 scaffolds is incomplete, hampering a comprehensive analysis of the genome. We have assembled the complete circular genome of a wAlbB strain from the Aa23 cell line, from long-read PacBio sequencing data at 450X coverage. The assembled circular chromosome is 1,484,007 bp in size, an increase of 321 kb over the published wAlbB genome, making it the largest sequenced Wolbachia genome to date. The annotation of the genome identified 1,207 protein coding genes, 34 tRNA, 3 rRNA and 1 tmRNA loci. The long reads enabled sequencing over complex repeat regions which have been be difficult to resolve with short-read sequencing. The availability of a complete circular genome from wAlbB will enable further biochemical, molecular and genetic analyses on this strain and related Wolbachia.
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Prediction of Golden Gate Assembly GGA Using a Comprehensive Analysis of T4 DNA Ligase End-Joining Fidelity and Bias (2018)
The one-pot assembly of long DNA sequences from multiple component parts is key to the rapid generation of constructs for modern synthetic biology. Methods for the one-pot assembly of multiple fragments linked by short overhangs (e.g. Golden Gate) depend on accurate and unbiased ligation. Design of junctions to date largely depends on the use of rules of thumb and empirical success, rather than detailed data on ligase fidelity and bias. In this study, we have applied Pacific Biosciences Single-Molecule Real- Time sequencing technology to directly measure of the ligation frequency of every possible 5′-four-base overhang pairing in a single experiment. This comprehensive data set has been applied to predict the accuracy of Golden Gate assembly (GGA) using the Type IIS restriction enzyme BsaI. Ten fragment assemblies were designed based on the ligation data with junctions predicted to result in high or low fidelity assembly. Experimental results confirmed not only the overall accuracy, but the specific mismatch ligation errors observed and their relative frequency. The data was further used to design 12- or 24- fragment assemblies of the lac operon, which were shown to assemble with high fidelity and efficiency. Thus, ligase fidelity data allows the prediction of high-accuracy overhang pair sets with greater flexibility in design than the rules of thumb, allowing assembly of >20 fragments at high-accuracy junction points even within defined coding regions without modification of the native DNA sequence.
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A novel enzyme isolated from a hot spring metagenomic DNA library defines a new CAZy family (GH154) of exosialidases with an inverting catalytic mechanism (2018)
Exosialidases (also termed neuraminidases; E.C. 3.2.1.18) are glycoside hydrolases that catalyze removal of a single terminal sialic acid from a subterminal sugar in an oligosaccharide. They are widely distributed in biology, having been found in prokaryotes, eukaryotes and certain viruses. Most characterized prokaryotic sialidases derive from organisms that are pathogenic or commensal with mammals. Less is known about sialidases from noncommensal microorganisms, including those that thrive in an extreme environmental niche like hypersaline ponds, evaporation salterns or thermal springs. In this study, we sought to explore if active sialidases could be identified from organisms that populate a thermal spring.
To address this question, we constructed a fosmid library in Escherichia coli from metagenomic DNA that had been isolated from green microbial mats from the Dixie Hot Spring in Nevada. A total of 616 E. coli clones, each having a fosmid with an insert of ~40 kb of environmental DNA, were created and arrayed in microtiter plates for screening. The library was screened for sialidases with two substrates: 2′-(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid (4MU-Neu5Ac) and 5-bromo-4-chloro-3-indolyl α-D-N-acetylneuraminic acid (X-Neu5Ac).
A single E. coli clone having sialidase activity was identified using both substrates. The fosmid was isolated from this strain, sequenced using the Pacific Biosciences DNA sequencing platform, and encoded ORFs were predicted with MetaGeneMark. The DNA sequence did not match any reported sequences from known microorganisms. Additionally, none of the predicted ORFs showed homology to existing sialidase families. Tn5 mutagenesis was conducted to identify the 505 amino acid ORF responsible for the enzymatic activity. BLASTP using the ORF’s deduced protein sequence analysis indicated that it was a member of a small family of bacterial “hypothetical” proteins with no known function. The protein was recombinantly over-expressed in E. coli and was shown to hydrolyze a variety of sialic acid containing substrates. Additionally, protein NMR showed that the enzyme functions via an inverting catalytic mechanism, a biochemical property distinct from known exosialidases that each function via a retaining mechanism. This unique inverting exosialidase defines a novel CAZy glycoside hydrolase family that has been designated GH154. -
Identification and Biosynthesis of Thymidine Hypermodifications in the Genomic DNA of Widespread Bacterial Viruses (2018)
Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their geneLc material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the DelPia phage ΦW-14 and the Bacillus phage SP10 contain the hypermodified pyrimidines α- putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2ʹ-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the Pseudomonas phage M6 and the Salmonella phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) Nucleic Acids Res 41:7635–7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy) methyluridine (5-NeOmdU) and 5-(2-aminoethyl)uridine (5-NedU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-NeOmdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation.
