PubMed · 6963414
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W de Oliveira, J V de Moraes, J E Junho de Araujo. 1982. [Artificial teeth].. https://pubmed.ncbi.nlm.nih.gov/6963414/
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Individual fluorescent molecules and individual singly labeled proteins were observed in the water-filled pores of poly(acrylamide) gels by far-field microscopy. Brownian motion was markedly reduced by the gel framework, thus enabling extended study of single fluorophores in aqueous environments. A highly axially dependent laser field was used both to excite the fluorophores and to image the molecules in three dimensions. Single molecules were followed as they moved within and through the porous gel structure. In contrast to dry polymeric hosts, these water-based gels may form a useful medium for single-molecule studies of biological systems in vitro.
The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solutions has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and separation conditions, and enhanced base-calling software. The influences of LPA molecular weight and concentration on separation were investigated. Additionally, the separation buffer, column temperature, and electric field were adjusted to increase the number of resolvable DNA fragments per run while maintaining an enhanced separation speed. Using low concentrations [2% (w/v)] of high molecular weight LPA polymers (> 5.5 x 10(6) Da), elevated column temperature (50 degrees C) and moderately high field (150 V/cm), rapid sequencing analysis for more than 1000 bases on a model ssM13mp18 template was obtained with 96.8% accuracy.
Capillary polymer sieving electrophoresis using dynamic sieving polymer solutions provides size-based separations of oligonucleotides. The polymer sieving system described here resolves single-stranded oligonucleotides with single-base resolution up to lengths of 30 bases within 10 min. The effect of temperature on the separation of synthetic oligonucleotide standards was examined between 20 degrees C and 40 degrees C, with optimal performance at 35-40 degrees C. By adding urea to the sieving buffer single-base resolution could be extended to 60 bases in about 40 min. Best performance was achieved with capillaries coated with a new monomer, acryloylaminoethoxyethanol. This coating provides the necessary stability to ensure long lifetimes.