Non-Enzymatic DNA Cleavage Reaction Induced by 5-Ethynyluracil in Methylamine Aqueous Solution and Application to DNA Concatenation

<div><p>DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5′-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.</p></div

Chemical structures of thymine (T) and 5-ethynyluracil (EU).

<p>Chemical structures of thymine (T) and 5-ethynyluracil (EU).</p

Chemical formula of the DNA cleavage reaction.

<p>R is expected to be an abasic sugar derivative.</p

Degradation of DNA oligonucleotides containing 5-ethynyluracil.

<p>(A), (B) HPLC charts of T₆(EU)T₆ before (gray) and after (black) the reaction in 14% NH₃aq (A) or 20% MeNH₂aq (B) at 70°C for 2 hours. (C), (D) (EU)T₂AT₂GT₂ (C) and T₂AT₂GT₂(EU)T (D) before (gray) and after (black) the reaction in 20% MeNH₂aq at 70°C for 2 hours.</p

Construction of plasmid from two PCR-amplified DNA fragments.

<p>(A) Scheme of plasmid construction. (B) Primer sequences used for PCR. The two sequences underlined in red and blue are complementary to each other. (C–G) Pictures of agarose gel electrophoresis. (C) PCR-amplified DNA fragments 1.5 (lane 2) and 2.2 kbp (lane 3). (D) 1.5 and 2.2 kbp DNA fragments before (lane 2,3) and after DNA cleavage at 25°C for 48 h (lane 4,5), 37°C for 10 h (lane 6,7), and 70°C for 0.5 h (lane 8,9). MeNH₂ was removed from the samples by speed-vac before electrophoresis. (E) Hybridized 1.5 and 2.2 kbp DNA fragments derived from those without cleavage reaction (lane 2) and cleaved at 25°C for 48 h (lane 3), 37°C for 10 h (lane 4), and 70°C for 0.5 h (lane 5). (F,G) Intact purified plasmids (F) and EcoRV-digested plasmids (G) derived from the DNA fragments cleaved at 25°C for 48 h (lane 2,3), 37°C for 10 h (lane 4–6), and 70°C for 0.5 h (lane 7–9). (H) Sequencing results of primer-derived regions of the plasmids. Underlined letters correspond to EU in the primers.</p

Schematic diagram of the key features of the global cDNA amplification method

<p><b>Copyright information:</b></p><p>Taken from "An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis"</p><p>Nucleic Acids Research 2006;34(5):e42-e42.</p><p>Published online 17 Mar 2006</p><p>PMCID:PMC1409679.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () Evaluation system to verify representation of amplified cDNA from diluted ES cellular RNA by Q-PCR and/or microarray. () Gene representation distorted during the global PCR. Diluted ES cellular RNA (10 pg) was amplified as described elsewhere (), and the replicates of amplification were sequentially sampled at 16, 20, 24, 28, 32, 36, 40 and 44 cycles. The expression levels of , , , , and were measured by Q-PCR, normalized by that of , and represented with brown, cyan, yellow, blue, pink and green lines, respectively. The averages of four independent experiments are plotted. () Schematic diagram of cDNA amplification. The mRNA and cDNA are colored pink and orange, respectively. The V1, V3 and T7 promoter sequences are represented by blue, red and green boxes, respectively. The bars above the letters represent the complementary sequences

Direct application of the newly developed method to single ICM cells from mouse E3

<p><b>Copyright information:</b></p><p>Taken from "An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis"</p><p>Nucleic Acids Research 2006;34(5):e42-e42.</p><p>Published online 17 Mar 2006</p><p>PMCID:PMC1409679.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p>5 blastocyst reveals the presence of two distinct cell populations. () Hierarchical clustering of single ICM cells. () Heat map representation of differentially expressed genes (top 100). The expression levels are color-coded from red (high) to blue (low). The expression levels are normalized in the lows. () The correlation of gene expression is preserved between E3.5 and E4.5. The copy numbers of expressed genes were estimated with Q-PCR. Orange, pink and green bars represent high, middle and low/non-detectable expression of , respectively. -values of the Chi-square test for independence from expression are indicated. (D and F) Blastocysts at E3.5 () and E4.5 (). The typical embryos used for single-cell experiments are shown. (E and G) Expression levels of key genes related to PE and epiblast at E3.5 () and E4.5 (). All of the single-cell samples of ICMs are shown. The representation code is the same as in (C)

Transcriptome Tomography for Brain Analysis in the Web-Accessible Anatomical Space

<div><p>Increased information on the encoded mammalian genome is expected to facilitate an integrated understanding of complex anatomical structure and function based on the knowledge of gene products. Determination of gene expression-anatomy associations is crucial for this understanding. To elicit the association in the three-dimensional (3D) space, we introduce a novel technique for comprehensive mapping of endogenous gene expression into a web-accessible standard space: Transcriptome Tomography. The technique is based on conjugation of sequential tissue-block sectioning, all fractions of which are used for molecular measurements of gene expression densities, and the block- face imaging, which are used for 3D reconstruction of the fractions. To generate a 3D map, tissues are serially sectioned in each of three orthogonal planes and the expression density data are mapped using a tomographic technique. This rapid and unbiased mapping technique using a relatively small number of original data points allows researchers to create their own expression maps in the broad anatomical context of the space. In the first instance we generated a dataset of 36,000 maps, reconstructed from data of 61 fractions measured with microarray, covering the whole mouse brain (ViBrism: <a href="http://vibrism.riken.jp/3dviewer/ex/index.html" target="_blank">http://vibrism.riken.jp/3dviewer/ex/index.html</a>) in one month. After computational estimation of the mapping accuracy we validated the dataset against existing data with respect to the expression location and density. To demonstrate the relevance of the framework, we showed disease related expression of Huntington’s disease gene and <i>Bdnf</i>. Our tomographic approach is applicable to analysis of any biological molecules derived from frozen tissues, organs and whole embryos, and the maps are spatially isotropic and well suited to the analysis in the standard space (e.g. Waxholm Space for brain-atlas databases). This will facilitate research creating and using open-standards for a molecular-based understanding of complex structures; and will contribute to new insights into a broad range of biological and medical questions.</p></div

Transcriptome Tomography.

<p>(<b>A</b>) <b>A schematic illustrated using a model material.</b> Two types of data, material shape images (drawn with green lines) and gene expression densities (shown in red) of fractions (indicated with asterisks), are obtained with sectioning, conjugated with block-face imaging and expression density measurement, along three body axes (shown in parentheses). The three series of sectioning are named after orthogonal planes (C, S and H). The densities are assigned to the voxels (pixels on a regular grid in a 3D space) in the images (as shown with +) and subjected to tomographic reconstruction (indicated in purple). A series of the process from one direction needs one material; therefore, at least three genetically identical materials were required. (<b>B</b>) <b>An outline of the technique and the first dataset creation.</b> Two types of data, fraction templates, which are the material shape image (in green) and fraction data, which are gene expression densities measured with microarray (in dotted red), were acquired from the same fractions prepared with a sectioning machine 3D-ISM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373-Yokota1" target="_blank">[12]</a>. The fractions were named “image fractions” for the former data and “material fractions” for the latter (the preparation process seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373.s007" target="_blank">Video S1</a>). Six fraction templates for the first dataset, two groups of three series sectioned in each of orthogonal and slightly oblique to the orthogonal planes: S/C/H and So/Co/Ho, composed of 9/13/6 and 10/16/7 fractions, respectively, (61 fractions in total as seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373.s001" target="_blank">Figure S1B</a>), are shown with fraction numbers in Template C: 13 fractions of 1 mm (5 µm×200 sections)-thickness. The pseudo-tomography technique of mapping in a single coordinate space (named ViBrism) including image registration, pseudo-back projection and tomographic reconstruction is shown in the flowchart (see details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373.s001" target="_blank">Figure S1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373.s005" target="_blank">Text S1</a>). After volume rendering, 3D expression maps for genes (a sample: Slitrk6) are visualized as pseudo-colored expression densities and anatomical images with an 80% cutoff filter (also seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373.s008" target="_blank">Video S2</a>). Slitrk6 is known to be expressed mostly in the thalamus as shown in the Allen Brain Atlas and BrainStars databases: 2Dand 3D views displayed here are compatible to those data shown below in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone-0045373-g004" target="_blank">Figure 4A and B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373.s008" target="_blank">VideoS2</a>.</p

Results for the computational experiment of reconstruction using 1,366 test spheres.

<p>Gene expression that was evenly distributed in one of the test spheres located randomly in the virtual brain of ViBrism was computationally reconstructed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373.s006" target="_blank">Text S2</a> for Supporting Methods). A histogram for the number of test spheres with true positive rates (% of TP: percentages of test sphere volumes overlapped with the reconstructed area) is shown. Maps of the reconstructed results (shown in yellow) with the test spheres (in red) are attached. In 2D maps, the 80% cutoff filter was applied to the results of left-upper S panels; otherwise, the reconstructed densities are shown in gray scales. 3D maps are shown with the filter. Approximately one fifth (20.4%) of the test spheres had more than 95% of TP, which is the mode in the histogram, and 94.7% in total had at least 5% of TP as indicated. One of the mode results, the median result (TP = 80%) and one of the poorly reconstructed results (TP<5%) are shown. Only 0.8% of the test spheres resulted in no TP, which was mainly due to the peripheral location of the test spheres in the virtual brain (data not shown).</p