PubMed · 8746683
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K D Altria. 1996. Troubleshooting.. https://doi.org/10.1385/0-89603-315-5%3A105
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An automated on-line ionic detergent removal pre-column system coupled to capillary liquid chromatography-electrospray mass spectrometry is described. The system involves two micro precolumns, composed of a specific ionic detergent trapping column and a preconcentration column, respectively, and a packed 300 microns I.D. analytical column. Sample loading to the micro precolumns and regeneration of the detergent trapping column were performed at a flow-rate of 50 microliters/min, while the flow-rate through the analytical column was set at 5.0 microliters/min. Ionic detergent-containing tryptic-digested protein samples were directly applied to the micro precolumns without sample pretreatment and were analysed by UV absorption detection and electrospray mass spectrometry. The presented system allows for the fully automated removal of SDS with virtually no loss in protein/peptides. Maximum SDS load and breakthrough have been determined. Excellent protein recovery and complete removal of SDS is found. The chromatographic separation after SDS removal was completely restored and equalled the reference chromatogram. Mass spectral data confirm these findings. Finally, this technique allows for SDS removal from minute protein samples without the need for any sample handling.
A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe's average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 micrograms unspecific DNA without post-PCR probe manipulations could be achieved with different primer/ probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.
A fluorescence-based, T7 (Sequenase) dye terminator method for sequencing double-stranded DNA using strings of three contiguous hexamers as primers and single-stranded binding protein is described. In this method, the circular, supercoiled DNA vector pUC19 is first linearized with a restriction enzyme to create a sequenceable template. Sequencing is then accomplished using three cycles of "denaturation," annealing, and extension/termination. Twenty-two of 33 hexamer strings tested in a controlled study produced acceptable sequence, with read lengths varying from the mid 300s to the low 400s and a base-calling accuracy of at least 97%. To test its potential utility in directed DNA sequencing, the protocol was then used to completely sequence both strands of pUC19. For this test project, a total of 28 hexamer strings was used with an overall successful priming rate of 75%. The current protocol appears to be sufficiently robust to be used in the finishing phase of a shotgun sequencing project and is amenable to semiautomation. Prospects for using the protocol for full-scale directed sequencing as well as for full automation are discussed.