Capillary gel electrophoresis of nucleic acids in pluronic F127 copolymer liquid crystals

AbstractIndustrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Capillary gel electrophoresis primarily separates analyte molecules based on their hydrodynamic volume to charge ratios, thus, offers excellent resolution for most of such otherwise difficult-to-separate isomers. In this work, two commercially available gel compositions were evaluated on the analysis of a mixture of ten synthetic HMOs. The relevant respective separation matrices were then applied to selected analytical in-process control examples. The conventionally used carbohydrate separation matrix was applied for the in-process analysis of bacteria-mediated production of 3-fucosyllactose, lacto-N-tetraose, and lacto-N-neotetraose. The other example showed the suitability of the method for the in vivo in-process control of a shake flask and fermentation approach of 2′-fucosyllactose production. In this latter instance, borate complexation was utilized to efficiently separate the 2′- and 3-fucosylated lactose positional isomers. In all instances, the analysis of the HMOs of interest required only a couple of minutes with high resolution and excellent migration time and peak area reproducibility (average RSD 0.26% and 3.56%, respectively), features representing high importance in food additive manufacturing in-process control. Graphical abstract

Download Full-text

Optimized Large Scale Synthesis of Phosphorothioate Oligonucleotides on PS-PEG Supports

Zeitschrift für Naturforschung B ◽

10.1515/znb-1997-0122 ◽

1997 ◽

Vol 52 (1) ◽

pp. 110-116

Author(s):

Michael Gerster ◽

Martin Maier ◽

Nils Clausen ◽

Jens Schewitz ◽

Ernst Bayer

Keyword(s):

Gel Electrophoresis ◽

Continuous Flow ◽

Large Scale ◽

Electrospray Mass Spectrometry ◽

Small Scale ◽

Batch Mode ◽

Crucial Step ◽

Capillary Gel Electrophoresis ◽

Phosphoramidite Chemistry ◽

Large Scale Synthesis

Sulphurization is a crucial step during synthesis of phosphorothioate oligonucleotides. Insufficient reaction leads to inhomogeneous products with phosphodiester defects and subsequently to destabilization of the oligomers in biological media. To achieve a maximum extent of sulphur incorporation, various sulphurizing agents have been investigated. Solely, the use of Beaucage reagent provided satisfactory results on PS-PEG supports. Based on our investigations in small scale synthesis (1 μmol) with continuous-flow technique, upscaling to the 0.1-0.25 mmolar range has been achieved using a peptide synthesizer. The syntheses were performed in batch mode with standard phosphoramidite chemistry. Additionally, large scale synthesis of a phosphodiester oligonucleotide has been carried out on PS-PEG with optimized protocols and compared to small scale synthesis on different supports. Products were analysed by 31P NMR, capillary gel electrophoresis and electrospray mass spectrometry. An extent of sulphurization of 99% and coupling effiencies of more than 99% were obtained and the products proved to have similar purity compared to small scale syntheses on CPG

Download Full-text

Properties of the single protein and two nucleic acids of tomato ring-spot virus

Canadian Journal of Botany ◽

10.1139/b77-120 ◽

1977 ◽

Vol 55 (8) ◽

pp. 1028-1037 ◽

Cited By ~ 8

Author(s):

Wayne R. Allen ◽

H. F. Dias

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Gel Electrophoresis ◽

Average Molecular Weight ◽

Virus Genome ◽

Protein Subunit ◽

Spot Virus ◽

Single Protein ◽

Middle Component ◽

Ring Spot

Purified preparations of several isolates of tomato ring-spot virus were shown by rate-zonal centrifugation in sucrose and equilibrium centrifugation in CsCl to be composed of two individual nucleoprotein components. Acrylamide-gel electrophoresis showed that the lighter (middle) component contained a nucleic acid (RNA 2) that was distinct from the species (RNA 1) contained in the heavier (bottom) component. The bottom was more infectious than the middle component and infectivity was enhanced by mixing the components, indicating that the virus genome is divided between component types. Similar results were obtained from infectivity tests on the two nucleic acids. The nucleic acid contents of the middle and bottom components were about 40 and 41%, respectively. The average molecular weights of RNA 2 and RNA 1 from three virus isolates, as determined by acrylamide-gel electrophoresis, were 2.5 and 2.6 × 106, respectively. Molecular complexing between the RNA species during electrophoresis was prevented with the use of formamide. The single protein subunit from the same three isolates had an average molecular weight of about 58 000. Serological comparisons of five tomato ring-spot isolates associated with diseases of fruit trees and grapevines indicated that only the grape yellow vein strain was antigenically distinct. These and other properties indicate that this virus is similar to other members of the nepovirus group.

Download Full-text