|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
46. PCR Product Size Measurement using MALDI Mass Spectrometry
G.B. Hurst, Y. Kim, K. Weaver, and
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has considerable potential as a technique for rapid and accurate analysis of PCR products. We are pursuing two specific end-uses of this technique: mapping mutant phenotypes to chromosome regions and determining the extent of chromosomal rearrangements that are targets for mutagenesis in collaboration with ORNL's Laboratory for Comparative and Functional Genomics and screening of endogenous bacterial to assess genetic potential for bioremediation, in collaboration with Mary Lidstrom at the University of Washington. Current capabilities include virtually routine analysis with near single-base resolution up to 100 bases, and the less routine ability to measure 200-mers or larger products. We are working to further extend this technique to larger PCR products (and similarly-sized DNA derived from other sources), as well as to develop schemes for scaling up the applicability of MALDI to larger numbers of samples.
The salts and buffers necessary as reagents for the PCR act as interferences for the MALDI process, and therefore must be removed prior to MALDI-MS analysis. To overcome this problem, we have developed a rapid reverse-phase method for purifying PCR products 1, and have demonstrated the parallel implementation of this procedure in a 96-well microtiter-format using a filter plate loaded with the appropriate reverse-phase resin. Manual implementation of the 96-well purification method, using an 8-channel pipettor and a vacuum manifold, requires approximately 20-30 minutes for 96 samples.
For MALDI-MS analysis, PCR products must be combined with a matrix material and allowed to dry on a sample plate. The resulting inhomogeneous spot is sparsely dotted with regions that yield useful spectra when interrogated with the desorption laser ("sweet spots"), and therefore requires either human expertise for laboriously choosing promising locations across the sample, or inefficient automated positioning of the laser at numerous positions in hopes of locating a sweet spot. For this reason, we are developing methods for preparing more homogeneous spots containing the mixture of PCR product and matrix, using polymeric substrates and/or additives. Fluorescently-labeled primers or PCR products may allow us to correlate MALDI-MS results with fluorescence microscopy imaging of the DNA distribution in these samples.
1 "MALDI-TOF Analysis of Polymerase Chain Reaction Products from Methanotrophic Bacteria," G.B. Hurst, K. Weaver, M.J. Doktycz, M.V. Buchanan, A.M. Costello, and M.E. Lidstrom, Anal. Chem. 70, 2693-2698 (1998).
Research supported by the Environmental Management Science Program, Office of Biological and Environmental Research, U.S. Department of Energy, and the Oak Ridge National Laboratory Director's Research and Development Funds. Oak Ridge National Laboratory is managed for the United States Department of Energy by Lockheed Martin Energy Research Corp. under contract DE-AC05-96OR22464.
|Author Index||Sequencing Technologies & Resources||Microbial Genome Program|
|Search||Mapping||Ethical, Legal, & Social Issues|