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  1. miRNA Detection by Ligation and Amplification of Complementary DNA Oligos Using SPlintR® Ligase (2015)

    Ligation of two adjacent DNA oligonucleotides splinted by RNA has historically been difficult to achieve. We have discovered that SplintR® ligase (Chlorella virus DNA ligase) is much more efficient in this ligation than T4 DNA ligase, which was traditionally used for this application. When these ligases were compared using the same RNA:DNA substrates the SplintR® enzyme achieved complete ligation with 10X less ligase and was 15X faster than T4 DNA ligase [1].

  2. Molecular Diagnostics for Gastrointestinal Parasites and Impact on Intestinal Microbiota in Rural Agentinian Children (2015)

    • 2 billion GI parasite infections worldwide
      • Poorest and resource-deprived communities
    • Standard method of diagnosis: Stool microscopy
      • Sensitivity variable depending on prevalence, species, and concentration method
        • 50-90% sensitivity
      • Underestimates polyparasitism
    • qPCR is rapid, quantitative, and high throughput species-specific method
    • GI parasites may disrupt normal intestinal microbiota
      • Decreased biodiversity is associated with disease
        • Malabsorption
        • Inflammatory bowel diseases

  3. In Vitro Reconstitution of Thermococcus Species 9°N Okazaki Fragment Maturation (2015)

    During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications and generation of doublestranded DNA breaks.To address the components required for the process, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed.

  4. One Ring To Bind Them All: Is HemeBiosynthesis A Factor In Wolbachia-Filarial Nematode Endosymbiosis? (2015)

    Transmitted by insect vectors, human nematode-based filariasis causes debilitating diseases affecting nearly 150 million people with 1.2 billion individuals at risk in 80 countries. Genomic sequencing has revealed that many human filarial nematodes, such as Wuchereria bancrofti, Brugia malayi and B. timori, (causative agents of lymphatic filariasis (LF)) and Onchocerca volvulus (causative agent of onchocerciasis (river blindness)) contain the obligate endosymbiont, Wolbachia. Genome sequencing of B. malayi (Bm) and its Wolbachia (wBm) identified a number of metabolites implicated in the host-endosymbiont interaction, one of which was heme, a co-factor in a number of enzymes and essential to many biological processes. Although the Bm genome encodes a functional ferrochelatase gene (the final step in the heme biosynthetic pathway and a product of lateral gene transfer), like other nematodes, it is incapable of synthesizing heme. However, the wBm genome contains a functional heme synthesis pathway, leading to the hypothesis that wBm may supply Bm with heme

  5. Improving Sample Workflow: Rapid PNGase F for Accurate Antibody Characterization (2015)

    The characterization of glycoprotein structure is becoming increasingly sophisticated, as regulatory agencies require multiple attributes to be measured during development, production, and formulation of biological drugs.

    Precise determination of N- and O-glycosylation, site occupancy, disulfide shuffling, misassembly, deamidation, oxidation, etc, require robust methods for sample preparation, to facilitate mass spectrometry analysis.

    Enzymes for glycan removal, along with specific proteases, are critical to these studies. Improved methods where glycosidases are combined, and/or coupled with labeling reactions or protease digestion, maximize reproducibility by eliminating handling errors. These methods, in turn, permit a more stringent definition of an original, biosimilar, or biobetter, facilitating formulation and process development innovations.

    We present in this poster new glycan removal protocols, including fast deglycosylation (using Rapid PNGase F) and deglycosylation of intact plant-derived glycoproteins (using PNGase Ar). These reactions were coupled with a simplified and versatile glycan labeling reaction by reductive amination, suitable for glycans lacking a glycosylamine end group.

    Also, glycosidase combinations were tested for complete N- and O-glycan removal, to facilitate proteomic analysis for glycoproteins that are heavily glycosylated.

    Finally, an enzyme mix containing PNGase F and Trypsin was used to prepare an IgG sample for peptide mapping. This abbreviated workflow maintained sensitivity and reproducibility

  6. Benefits of Illumina® Library Quantitation with NEBNext® Library Quant Kit (2015)

    Accurate quantitation of a next-generation sequencing library is essential to maximizing data output and quality from each instrument run. qPCR is widely accepted as the most effective method for library quantitation by both users and manufacturers, as qPCR methods measure only sequenceable library fragments with a high level of accuracy and consistency. The NEBNext Library Quant Kit for Illumina presents a simple, robust method for quantitation of Illumina libraries. Here we demonstrate the effectiveness of the Kit for a broad range of library types and sizes as well as advantages offered by qPCR quantitation for obtaining optimal cluster density and user-to-user consistency. The NEBNext Quant Kit offers an efficient and cost-effective qPCR library quantitation workflow for users looking to optimize both sequencing yield and throughput.

  7. Crystal Structure of the 8 bp-Specific Restriction Enzyme SwaI (2015)

    SwaI, a Type IIP restriction enzyme from Staphylococcus warneri cleaves the symmetric sequence ATTT|AAAT, producing fragments with blunt ends (‘|’ = cleavage site). We solved the crystal structure of SwaI alone, of SwaI bound to uncleaved DNA in the presence of Ca2+ ions, and of SwaI bound to cleaved DNA in the presence of Mg2+ ions. We describe these structures, and compare them to that of PacI, which cleaves the related 8-bp sequence, TTAAT|TAA.

  8. Small RNA-Mediated DNA Methyltransferase 1 Inhibition Causes DNA Methylation Alteration in Mammalian Cells (2015)

    Uses hemimethylated DNA as substrate.

    • Responsible for CpG methylation maintenance.
    • Dysregulation of DNMT1 causes aberrant methylation in cancer.
    • Highly abundant transcripts inhibit DNMT1 activity in vivo (asCEBPA).
    • LncRNA (Kcnq1ot1) recruits DNMT1 to imprinted genes and somatic DMR.

  9. NiCo21(DE3): A BL21(DE3) Derivative Designed for Expression and Purification of His-tagged Recombinant Protein (2015)

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly co-eluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA and optionally AceE fused at their C-terminus to a chitin binding domain (CBD), and the protein GlmS with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flow-through step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

  10. Complete Genome Sequence and Methylome Analysis of Beggiatoa leptomitoformis strains D401 and D402

    The taxonomy of Beggiatoa genus is still a work in progress. Despite many morphotypes of the Beggiatoa genus having been described in the literature, only one species, B. alba has been validated until now. In 2016, we described a second species, B. leptomitoformis. Two strains of B. leptomitoformis D401 and D402 have been isolated from different regions of Russia. They differ in their morphology and physiology especially their ability to growth lithotrophically in the presence of thiosulfate.  While B. leptomitoformis D402 is able to accumulate elemental sulfur, strainD401 cannot.  We performed genomic sequencing of these two strains using the PacBio SMRT platform and assembled the reads into two complete circular genomes: B. leptomitiformis D-401 with 4,266,286 bp and D-402 with 4,265,296 bp. Both genome sequences have been deposited in GenBank with accession numbers CP018889 and CP012373 respectively (1). Surprisingly these two genomes showed almost 99% identity. The preliminary analysis of several thiosulfate oxidation (,soxA, soxF and soxW) and autotrophic  assimilation of CO2 (cbbQ, rbcL) metabolic operones in both stains did not revel any noticeable differences.

    The advantage of the PacBio sequencing platform is its ability to detect the epigenetic state of the sequenced DNA, which allows for the identification of modified nucleotides and the corresponding motifs in which they occur. Thirteen DNA methyltransferase recognition motifs were found. They include one m4C and nine m6A modifications that were detected by direct SMRT sequencing and an additional three m5C motifs were detected in Tet2 treated DNA. The motifs were then matched with methyltransferase genes in the genome, and the results have been deposited in REBASE (2).

    (1) Fomenkov,A., Vincze, T., Grabovich, M.Y., Dubinina, G., Orlova, M., Belousova, E., Roberts, R.J. (2015)  Complete Genome Sequence of the Freshwater Colorless Sulfur Bacterium Beggiatoa leptomitiformis Neotype Strain D-402. Genome Announc., 6:e01436. DOI:10.1128/genomeA.01436-15

    (2) Roberts, R.J., Vincze, T., Posfai, J., Macelis, D. REBASE-a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res. 43: D298-D299 (2015). DOI: 10.1093/nar/gku1046

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