Nucleotide Discrimination with DNA Immobilized in the MspA Nanopore

Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices

The NTD Nanoscope: potential applications and implementations

<p>Abstract</p> <p>Background</p> <p>Nanopore transduction detection (NTD) offers prospects for a number of highly sensitive and discriminative applications, including: (i) single nucleotide polymorphism (SNP) detection; (ii) targeted DNA re-sequencing; (iii) protein isoform assaying; and (iv) biosensing via antibody or aptamer coupled molecules. Nanopore event transduction involves single-molecule biophysics, engineered information flows, and nanopore cheminformatics. The NTD Nanoscope has seen limited use in the scientific community, however, due to lack of information about potential applications, and lack of availability for the device itself. Meta Logos Inc. is developing both pre-packaged device platforms and component-level (unassembled) kit platforms (the latter described here). In both cases a lipid bi-layer workstation is first established, then augmentations and operational protocols are provided to have a nanopore transduction detector. In this paper we provide an overview of the NTD Nanoscope applications and implementations. The NTD Nanoscope Kit, in particular, is a component-level reproduction of the standard NTD device used in previous research papers.</p> <p>Results</p> <p>The NTD Nanoscope method is shown to functionalize a single nanopore with a channel current modulator that is designed to transduce events, such as binding to a specific target. To expedite set-up in new lab settings, the calibration and troubleshooting for the NTD Nanoscope kit components and signal processing software, the NTD Nanoscope Kit, is designed to include a set of test buffers and control molecules based on experiments described in previous NTD papers (the model systems briefly described in what follows). The description of the Server-interfacing for advanced signal processing support is also briefly mentioned.</p> <p>Conclusions</p> <p>SNP assaying, SNP discovery, DNA sequencing and RNA-seq methods are typically limited by the accuracy of the error rate of the enzymes involved, such as methods involving the polymerase chain reaction (PCR) enzyme. The NTD Nanoscope offers a means to obtain higher accuracy as it is a single-molecule method that does not inherently involve use of enzymes, using a functionalized nanopore instead.</p

Alteration of gene expression by alcohol exposure at early neurulation

<p>Abstract</p> <p>Background</p> <p>We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables.</p> <p>Result</p> <p>Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, <it>Sox5, Bhlhe22</it>), neural growth factor genes [<it>Igf1, Efemp1</it>, <it>Klf10 </it>(<it>Tieg), and Edil3</it>], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (<it>Rbp1</it>), and <it>de novo </it>expression of aldehyde dehydrogenase 1B1 (<it>Aldh1B1</it>). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos.</p> <p>Conclusion</p> <p>This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.</p

The Potential and Challenges of Nanopore Sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of ‘third generation’ instruments that will sequence a diploid mammalian genome for ~$1,000 in ~24 h.Molecular and Cellular BiologyPhysic

The properties and applications of single-molecule DNA sequencing

Single-molecule sequencing enables DNA or RNA to be sequenced directly from biological samples, making it well-suited for diagnostic and clinical applications. Here we review the properties and applications of this rapidly evolving and promising technology

Microdissection of Shoot Meristem Functional Domains

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize