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Rapid mass spectrometric DNA diagnostics for assessing microbial community activity during bioremediation. 1997 annual progress report

'The effort of the past year''s activities, which covers the first year of the project, was directed at developing DNA-based diagnostic procedures for implementation in high through-put analytical instrumentation. The diagnostic procedures under evaluation are designed to identify specific genes in soil microorganisms that code for pollutant-degrading enzymes. Current DNA-based diagnostic procedures, such as the ligase chain reaction (LCR) and the polymerase chain reaction (PCR), rely on gel electrophoresis as a way to score a diagnostic test. The authors are attempting to implement time-of-flight (TOF) mass spectrometry as a replacement for gel separations because of its speed advantage and potential for sample automation. The authors anticipate that if TOF techniques can be implemented in the procedures, then a very large number of microorganisms and soil samples can be screened for the presence of specific pollutant-degrading genes. The use of DNA-based procedures for the detection of biodegrading organisms or genes that code for pollutant-degrading enzymes constitutes a critical technology for following biochemical transformation and substantiating the impact of bioremediation. DNA-based technology has been demonstrated to be a sensitive technique for tracking micro-organism activity at the molecular level. These procedures can be tuned to identify groups of organisms, specific organisms, and activity at the molecular level. They are developing a P-monitoring strategy that relies on the combined use of DNA diagnostics with mass spectrometry as the detection scheme. The intent of this work is a two-fold evaluation of (1) the feasibility of replacing the use of gel separations for identifying polymerase chain reaction (PCR) products with a rapid and automatable form of electrospray mass spectrometry and (2) the use of matrix-assisted-laser-desorption-ionization mass spectrometry (MALDI-MS) as a tool to score oligonucleotide ligation assays (OLA).'