Biomedical subjects

E Brassard

Publications and source records attributed to E Brassard.

5 recordsLinked to original sources

PubMed1992

Pulsed field sequencing gel electrophoresis.

The effect of pulsed fields on sequencing gel electrophoresis is investigated, using DNA fragment markers ranging in size from 20 to 6557 bases. For high continuous electric fields (5000 V/55 cm) band inversion is observed in which fragments larger than 4000 bases migrate faster than those of 800-1000 bases. The use of one-dimensional pulsed field gel electrophoresis (ODPFGE) eliminates band inversion and extends the monotonic size-mobility relationship of the DNA markers up to about 4000 bases. The relevance of these results, obtained using a manual sequencing process with autoradiographic detection, to automated sequences is discussed.

Autoanalysis↗

PubMed1991

Observation of orientation and relaxation of protein-sodium dodecyl sulfate complexes during pulsed intermittent field polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis (PAGE) of proteins denatured with SDS (sodium dodecyl sulfate) has been used successfully to separate proteins according to their molecular mass. In spite of the extensive use of this technique, the motion of the protein-SDS complex in a polyacrylamide gel is still not understood. Here we report on the observation of the orientation (in the field direction) and relaxation of protein-SDS complexes during pulsed intermittent field PAGE experiments. The results give an indication of the stiffness of the molecules and may be useful for the development of a technique to improve the separation of large proteins using pulsed electric fields.

Chemical Phenomena↗

PubMed1990

Molecular detrapping and band narrowing with high frequency modulation of pulsed field electrophoresis.

In high electric fields, megabase DNA fragments are found to be trapped, i.e. to enter or migrate in the gel only very slowly, if at all, leading to very broad electrophoretic bands and loss of separation. As a consequence, low electric fields are usually used to separate these molecules by pulsed field electrophoretic methods. We report here that high-frequency pulses eliminate the molecular trapping found in continuous fields. When high frequency pulses are used to modulate the longer pulses used in pulsed field electrophoresis, narrower bands result, and higher fields can be used. We suggest that this is due to effects that occur on the length scale of a single pore.

Candida↗

PubMed1990

Optimization of a polypyrrole glucose oxidase biosensor.

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of +0.65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentiostating the enzyme electrode at a potential of +0.7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determine the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0.3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0.01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0.17 microns (75 mC/cm2) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1.0 mM to 7.5 mM glucose and kinetic parameters determined for the optimized biosensors were 33.4 mM for the Km and 7.2 microA for the Imax. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.

Biosensing Techniques↗

PubMedDate not supplied

Manual sequencing using pulsed field.

The use of pulsed fields in manual sequencing opens up the compression zone found with a DC field and extends the range of resolution from a few hundred bases to several thousand bases. The band inversion problem is overcome with the proper pulsing conditions, and the bands are sharper than for the DC field case. Accurate visual reading is possible up to about 800-900 bases. The method is compatible with automation techniques, since the band spectrum is stretched continuously during migration, and the smaller fragments are run off the gel.

Base Sequence↗