Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

<div><p>Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.</p></div

Schematic of the building blocks for DNA construction.

<p>Streptavidin-coated magnetic beads were used as solid support for dsDNA synthesis and oligo fragments were ligated to the building block one at a time.</p

Schematic diagram for the proposed dsDNA synthesis.

<p>The overall procedure for dsDNA synthesis is composed of three processes: annealing, binding of streptavidin coated magnetic beads to biotinylated oligos, and ligation.</p

Agarose gel electrophoresis of annealed and ligation products.

<p>Lane 1 and 8, ladder; Lane 2, annealed product of N1b and N2b; Lane 3, annealed product of N3b and N4b; Lane 4, annealed product of N5b and N6b; Lane 5, “one-pot” ligation product of [(N1b-N2b)+(N3b-N4b)+(N5b-N6b)]; Lane 6, sequential ligation product of [(N1b-N2b)+(N3b-N4b)]+(N5b-N6b); Lane 7, ligation product of [(N1b-N2b)+(N5b-N6b)].</p

Sequence of the oligo fragments used to form the complete dsDNA.

<p>Sequence of the oligo fragments used to form the complete dsDNA.</p

Analysis of Global Gene Expression in <i>Brachypodium distachyon</i> Reveals Extensive Network Plasticity in Response to Abiotic Stress

<div><p><i>Brachypodium distachyon</i> is a close relative of many important cereal crops. Abiotic stress tolerance has a significant impact on productivity of agriculturally important food and feedstock crops. Analysis of the transcriptome of <i>Brachypodium</i> after chilling, high-salinity, drought, and heat stresses revealed diverse differential expression of many transcripts. Weighted Gene Co-Expression Network Analysis revealed 22 distinct gene modules with specific profiles of expression under each stress. Promoter analysis implicated short DNA sequences directly upstream of module members in the regulation of 21 of 22 modules. Functional analysis of module members revealed enrichment in functional terms for 10 of 22 network modules. Analysis of condition-specific correlations between differentially expressed gene pairs revealed extensive plasticity in the expression relationships of gene pairs. Photosynthesis, cell cycle, and cell wall expression modules were down-regulated by all abiotic stresses. Modules which were up-regulated by each abiotic stress fell into diverse and unique gene ontology GO categories. This study provides genomics resources and improves our understanding of abiotic stress responses of <i>Brachypodium</i>.</p></div

Differential expression of <i>Brachypodium distachyon</i> genes in response to stress.

<p><b>A</b>. Numbers of genes up-regulated (light grey bars) and down-regulated (dark grey) are shown as a function of time in hours after stress onset. <b>B</b>. Heatmap of expression differences between control and indicated stress arrays. Similar expression profiles are clustered in the dendrogram. Positive (green) and negative (red) differences between stress and control arrays are shown for all genes called as differentially expressed by SAM analysis. Columns are time points. Expression values are saturated at +/− 4 RMA, for display purposes. <b>C.</b> Venn diagram showing overlap of up-regulated genes in response to the four assayed abiotic stresses: cold (blue), heat (yellow), drought (purple) and salt (green). Area of overlaps is not proportional to the overlap. The numbers of genes in each region of the diagram are indicated. <b>D.</b> Venn diagram depicting intersections of sets of down-regulated genes in response to the four assayed abiotic stresses.</p