PubMed · 8977591
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P S Burge. 1996. Calibrating the calibrators.. https://doi.org/10.1136/thx.51.10.969
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Mass spectrometry has become an increasingly important tool of high accuracy, efficiency, and speed for the routine analysis of nucleic acids. To make it useful for large-scale sequencing of genomic material as required for example in genotyping and clinical diagnosis, it is necessary to find approaches that allow the analysis of sequences much larger than the 100 nucleotides currently possible. Matrix-assisted laser desorption/ionization (MALDI) mass spectra of synthetic DNA, restriction enzyme fragments of plasmid DNA, and RNA transcripts up to a size of 2180 nucleotides are reported. The demonstrated mass accuracy of 1 percent or better and the sample requirement of a few femtomoles or less surpass all currently available techniques for the analysis of large nucleic acids. DNA and RNA can be analyzed with only a limited investment in sample purification.
Applications of polymerase chain reaction (PCR) product analysis using rapid affinity capture followed by delayed extraction (DE) MALDI-TOFMS is presented. Such applications include multiplex short tandem repeat (STR) typing, which is demonstrated for STR systems from conventional and microchip-based thermal cycling instruments. Using the combination of the microfabricated PCR instrument and DE-MALDI-TOFMS, a complete genotyping assay can be performed in under 50 min with a resultant molecular weight accuracy approaching or exceeding 100 ppm through external calibration. The observed resolution and mass accuracy for a 69-base PCR product enables identification of single base substitutions by direct molecular weight determination.
A simple, accurate, precise, and versatile high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of three quinolone antibodies in Mueller-Hinton broth. The fluoroquinolone agents studied were ciprofloxacin, ofloxacin, and sparfloxacin; other quinolone agents have been identified using this method but not validated in this matrix (levofloxacin, clinafloxacin, temafloxacin, and trovafloxacin). In addition, several other biological growth mediums have been investigated (human serum, human urine, Todd-Hewitt growth media, Ensure enteral feeding solution, and Haemophilus growth media). This method uses UV detection (280 nm), a simple, one-step protein precipitation extraction, and separation using a C18 column with an isocratic, ion-pairing mobile phase. An appropriate internal standard was obtained by using another quinolone antibiotic of differing retention time. The calibration curves were linear (r2> or =0.999) over a concentration range of 0.0625-20.0 microg/ml with a lower limit of quantification of 0.1 microg/ml. The intra-day and inter-day coefficients of variation were less than 15%.