V Murugaiah | PubMed Health

PubMed1997

Electrophore mass tag dideoxy DNA sequencing.

Toward a goal of dideoxy sequencing DNA utilizing electrophore labels, we prepared four electrophore-labeled DNA oligonucleotide primers. Each primer has a different electrophore and DNA sequence but a common glycol keto (alpha,beta-dihydroxyketo) release group. Cleavage of this latter group by either periodate oxidation or a thermal retroaldol reaction releases the electrophores for detection by mass spectrometry. Successful sequencing data with these primers was obtained by capillary electrophoresis on an ABI Model 310 after fluorescence dideoxy terminator cycle sequencing reactions were conducted. In a separate experiment, it was demonstrated that a cocktail of the four electrophore DNA primers could be detected as a dried sample spot by CO2 laser desorption/capillary collection/gas chromatography electron capture mass spectrometry. These results establish some feasibility for our long-term goal of high-speed multiplex electrophore mass tag dideoxy DNA sequencing. Ultimately we plan to use a higher number of electrophore mass tags and to rely on direct detection of the desorbed electrophores by electron capture time-of-flight mass spectrometry.

Chromatography, High Pressure Liquid↗

PubMed1995

Masking as a mechanism for evaporative loss of trace analyte, especially after solid-phase extraction.

Using a Pasteur pipette plugged with silanized glass wool and packed with C18-silica particles, we attempted to remove K2CO3 from an aqueous acetonitrile solution. In spite of extensive washing of the column with water after the sample was applied, elution with acetonitrile followed by evaporation gave a visible, white residue. It was found that the residue was derived from both the sample and the packing, including particles from the latter. Substitution of a plastic column/polyethylene frit for the Pasteur pipette/glass wool gave a more consistent residue, apparently because this improved the retention of particles. Subsequent experiments were conducted in the plastic hardware. The amount of the residue was observed to vary as much as 19-fold when C18-silica particles were tested from different manufacturers, and the residue could be reduced in amount as much as 9-fold when a column was prepared in the laboratory vs. the use of a comparable, pre-packed column. The water itself contributed some of the residue: even the "purest" water routinely available left a visible residue when 1.0 ml was appropriately evaporated (e.g. on Saran Wrap in a microwave oven). The recovery of an arbitrary trace analyte and internal standard (pentafluorobenzylated nucleobases at the low pg level) was 32% less when they were evaporated in acetonitrile that had been passed through an acetonitrile and water-washed cartridge containing C18-Si vs. evaporation in untreated acetonitrile. Collectively these results reveal that an evaporation can risk some loss of an analyte from masking by even subtle solvent contaminants.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetonitriles↗