UV–visible absorption spectra (Ab) and fluorescence emission spectra (Em) of and

<p><b>Copyright information:</b></p><p>Taken from "Novel cyanine-AMP conjugates for efficient 5′ RNA fluorescent labeling by one-step transcription and replacement of [γ-P]ATP in RNA structural investigation"</p><p>Nucleic Acids Research 2005;33(4):e37-e37.</p><p>Published online 24 Feb 2005</p><p>PMCID:PMC549576.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The spectra were measured in 20 mM phosphate buffer, pH 7.0. All spectra were normalized to 1 at their λ. The λ difference between excitation and emission is 20 nm for both and

Colonies from 2-fragment assembly of varying lengths of overlapping nucleotides (0–25 nt OL) to construct the pGFP plasmid.

<p>The zero background can be seen from 0 and 6 nt OL. Increasing the OL size generates a higher number of colonies.</p

Correlation of plasmid size and the number of colonies.

<p>(A) The relationship between the number of colonies and plasmid size through 2-fragment assembly with 18 nt OL and 25 nt OL. (B) The relationship between the number of colonies and plasmid size from 3-fragment assembly with 18 nt OL and 25 nt OL. The error bar represents the range of variation.</p

<i>In vivo</i> cloning of up to 16 kb plasmids in <i>E</i>. <i>coli</i> is as simple as PCR

<div><p>The precise assembly of defined DNA sequences into plasmids is an essential task in bioscience research. While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning techniques based on restriction digestion and ligation are still commonly used in routine DNA cloning. Here, we describe a simple, fast, and economical cloning method based on RecA- and RecET-independent <i>in vivo</i> recombination of DNA fragments with overlapping ends using <i>E</i>. <i>coli</i>. All DNA fragments were prepared by a 2-consecutive PCR procedure with Q5 DNA polymerase and used directly for transformation resulting in 95% cloning accuracy and zero background from parental template plasmids. Quantitative relationships were established between cloning efficiency and three factors–the length of overlapping nucleotides, the number of DNA fragments, and the size of target plasmids–which can provide general guidance for selecting <i>in vivo</i> cloning parameters. The method may be used to accurately assemble up to 5 DNA fragments with 25 nt overlapping ends into relatively small plasmids, and 3 DNA fragments into plasmids up to 16 kb in size. The whole cloning procedure may be completed within 2 days by a researcher with little training in cloning. The combination of high accuracy and zero background eliminates the need for screening a large number of colonies. The method requires no enzymes other than Q5 DNA polymerase, has no sequence restriction, is highly reliable, and represents one of the simplest, fastest, and cheapest cloning techniques available. Our method is particularly suitable for common cloning tasks in the lab where the primary goal is to quickly generate a plasmid with a pre-defined sequence at low costs.</p></div

Confirmation of correct plasmid assembly by digestion with restriction enzymes.

<p>(<b>A</b>) pIkB digestion by SalI & NdeI resulting in 3 expected bands at 842, 2508, and 2979 bp, (<b>B</b>) pDcEG digestion by NsiI leading to 4 expected bands at 266, 1611, 2996, and 6940 bp, and (<b>C</b>) pDSADE digestion by NsiI generating 5 expected bands at 266, 1751, 2996, 4048, and 6940 bp. The 266 bp band was barely visible due to its expected weak intensity relative to other bands (only 3.8% of the 6940 bp band). No effort was made to identify the single negative plasmid (<b>lane 4</b> in <b>C</b>), but it was about 2.8 kb and contained KanR (the presence of the 266 bp signature band by NsiI digestion) as expected for growth under kanamycin conditions. It was not one of the two parental template plasmids (pDcEG & pDSA, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183974#pone.0183974.s004" target="_blank">S4 Table</a>) used for PCR.</p

The correlation between the number of colonies and the number of DNA fragments (2–5) with 18 nt OL and 25 nt OL to construct pGFP.

<p>For both 18 and 25 nt OL assemblies, the colony number rapidly decreases with the number of DNA fragments. The error bar represents the range of variation.</p

Colonies from <i>in vivo</i> assembly of 3–5 overlapping DNA fragments with 18 nt OL and 25 nt OL to construct pGFP.

<p>While increasing the OL size produces more colonies, the number of colonies decreases with increasing number of DNA fragments.</p

The relationship between cloning efficiency and the length of overlapping ends.

<p>(<b>A</b>) Identification of positive and negative colonies by fluorescence. The circled colonies were non-fluorescent, which were used to calculate cloning accuracy of plasmid assembly by <i>E</i>. <i>coli in vivo</i> recombination. (<b>B</b>) The relationship between the number of colonies and overlapping nucleotides through 2-fragment assembly to construct pGFP. The data were from 2–6 independent experiments for each point, and the error bar represents the range of variation. (<b>C</b>) High cloning accuracy of pGFP 2-fragment assembly, regardless of the length of overlapping nucleotides. The 9 nt OL point had apparent 100% accuracy and no variation from 2 independent experiments, but it only had 7 colonies in total.</p

CHIKV_mos infectivity can be enhanced after replication in mammalian cells.

<p>The viral stocks of CHIKV_vero-NIH3T3 and CHIKV_mos-NIH3T3 were prepared after CHIKV_vero or CHIKV_mos (Ross strain, MOI = 1) replicated in NIH3T3 for 48 h. (A) NIH3T3 and HFF cells (B) were infected with CHIKV_vero, CHIKV_mos, CHIKV_vero-NIH3T3 and CHIKV_mos-NIH3T3 (MOI = 1), and viral RNA copy numbers were measured at 24 h by RT-qPCR. Data represents ratio of copy number of CHIKV <i>E1</i> to <i>β-actin</i>. (C) NIH3T3 cells were infected with CHIKV_vero-L929 and CHIKV_mos-L929 (MOI = 1), and viral RNA copy numbers were measured by RT-qPCR at 24 h. (D) NIH3T3 cells were infected with CHIKV_vero-VERO and CHIKV_mos-VERO (MOI = 1), and viral RNA copy numbers were measured at 24 h by RT-qPCR. (E) Attachment of CHIKV_vero-NIH and CHIKV_mos-NIH (MOI = 1) were performed in NIH3T3 cells at 4°C for 1 h and attached viruses were measured by RT-qPCR. (F) GAGs neutralization of CHIKV_vero-NIH3T3 and CHIKV_mos-NIH3T3 attachment was performed with heparin (1000 U/mL), chondroitin sulfate A (CSA, 1000 μg/mL) and dermatan sulfate (DS, 1000 μg/mL) in NIH3T3 cells, and the attached viruses were quantified by RT-qPCR and normalized to the respective untreated controls. All data sets represent two independent experiments performed in triplicates. GAG-treated samples were compared to the respective controls (without GAGs) and analyzed using a one-way ANOVA (**** denotes <i>p <</i> 0.0001, *** denotes <i>p <</i> 0.0005, ** denotes <i>p <</i> 0.005, and <i>ns</i> denotes <i>p</i> ≥ 0.05).</p

CHIKV_mos has reduced attachment to host cells than CHIKV_vero.

<p><b>(</b>A), Equal PFUs of CHIKV_vero or CHIKV_mos (Ross strain) were added to Vero cells (left), NIH3T3 cells (middle) and L929 cells (right) for plaque development. (B), Plaque counts of CHIKV_vero and CHIKV_mos in the indicated cells were quantified. (C) L929, NIH3T3 and HFF cells were inoculated with CHIKV_vero or CHIKV_mos (Ross strain, MOI = 1) at 4°C for 1 h and attached viruses were quantified by RT-qPCR and presented as the ratio of CHIKV <i>E1</i> copy number per 1000 copy of cellular <i>β-actin</i>. (D) L929, NIH3T3 and HFF cells were inoculated with CHIKV_vero or CHIKV_mos (LR OPY1, MOI = 1) at 4°C for 1 h and viruses attached to cells were quantified by RT-qPCR. (E) NIH3T3 cells were infected with CHIKV_vero or CHIKV_mos (Ross strain, MOI = 2.5) at 4°C for 45 min and the virus-bound cells were quantified by flow cytometry. (F) CHIKV_vero or CHIKV_mos (100 PFUs) were added to monolayer of L929 cells at 4°C for 1 h and both attached and unattached viruses were quantified by plaque assay to calculate percentage attachment. (G) CHIKV_vero or CHIKV_mos (Ross strain, 100 PFUs) were added to the L929 cell monolayer at 4°C for 1 h. After removing unattached virus, cells were replaced with fresh medium (control), or treated with acidic medium (pH 5.5) for 2 minutes before adding fresh medium, or replaced with NH₄Cl (20 mM) containing media. Viruses that entered into cells were analyzed by allowing plaque development for 48 h and normalized to controls. (H) Viral RNA copies in L929 cells infected with CHIKV_vero or CHIKV_mos (Ross strain, MOI = 1) with or without NH₄Cl (20 mM) were measured by RT-qPCR at 24 h.p.i. Data represent at least two independent experiments performed in triplicates with the similar results. ND denotes not detected, and <i>ns</i> denotes not significant (<i>p</i> ≥ 0.05).</p