Biomedical subjects

S Mouradian

Publications and source records attributed to S Mouradian.

3 recordsLinked to original sources

PubMed1997

DNA analysis using an electrospray scanning mobility particle sizer.

A scanning mobility particle sizer (SMPS) allows size separation of gas phase particles according to their electrophoretic mobilities. The addition of an electrospray source (ES) recently allowed extension of SMPS analysis to the macromolecular range. We demonstrate here the application of ES-SMPS to nucleic acids analysis. Single- and double-stranded DNA molecules ranging from 6.1 kDa (single-stranded DNA 20 nucleotides in length) to 300 kDa (500 base-pair double-stranded DNA) were separated and detected by ES-SMPS at the picomole to femtomole levels. The measured electrophoretic mobility diameters were found to correlate with the analytes' molecular weights, while the peak areas could yield quantitative information. No fragmentation of DNA was observed under the conditions employed. Different apparent densities were observed for single-stranded and double-stranded DNAs, showing a different behavior for each type of biomolecule. The total analysis time was about 3 min/spectrum. Further optimization of ES-SMPS is expected to make it a fast and sensitive technique for biopolymer characterization.

DNA↗

PubMed1996

Analyzing sequencing reactions from bacteriophage M13 by matrix-assisted laser desorption/ionization mass spectrometry.

The current demand for improved DNA sequencing methodologies posed by the Human Genome Project has spurred the investigation of alternatives to gel electrophoresis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has great potential for the rapid analysis of DNA fragments. Mock Sanger sequencing mixtures have been successfully analyzed by MALDI by pooling synthesized oligonucleotides corresponding to the M13 bacteriophage sequence. More recently, analyses of Sanger sequencing fragments enzymatically generated from synthetic templates of 45 or 50 bases were reported. In the present study, these feasibility demonstrations are extended to show MALDI sequencing from the M13 bacteriophage DNA template commonly used in actual Sanger sequencing. The results show sequence determination for extension products up to 35 bases in length. Different desalting and purification procedures were investigated and it was found that salt could be efficiently reduced by removal of the template in a post-reaction step. Work in progress to stabilize DNA by chemical modification, employed in conjunction with the methods described here, should enable significant extension of the length of readable sequence.

Bacteriophage M13↗

PubMedDate not supplied

Separating field strength, temperature, and pulsing effects in pulsed field electrophoresis.

The utility of pulsed field electrophoresis for DNA sequencing is investigated. Previous studies have indicated a beneficial retardation of sequencing fragments when pulsed fields are employed. The interpretation of these results is complicated, however, by concomitant variations in electric field strength and/or temperature. Methods are presented here permitting discrimination of such mobility effects due to pulsing, field strength, and temperature. It is demonstrated that under the conditions employed here, observed mobility effects are due to electric field variations rather than pulsing. These conditions thus correspond to the low frequency/small molecule limit. The effect of temperature is estimated from the steady state solution to the heat conduction equation under appropriate boundary conditions. No temperature effect upon mobility is operative in the thin gel system employed, due to the high efficiency of heat transfer. However, it is shown that in conventional gel systems large temperature-related mobility effects can occur. These methods for dissecting and understanding mobility effects in pulsed field electrophoresis are expected to be of general utility.

Base Sequence↗