Gregory B. Hurst, Mitchel J. Doktycz[1], R.P. Woychik[2], Michelle V. Buchanan
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge TN 37831.
The polymerase chain reaction (PCR) produces many copies of a targeted DNA sequence, with information encoded in the size of the targeted sequence. The currently-accepted method for determining PCR product sizes is by gel electrophoresis, a lengthy and labor-intensive process that is prone to inaccuracies. As a potentially faster and more accurate alternative to gel electrophoresis, we are developing mass spectrometric methods based on matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS) for measuring PCR product molecular weights. Because the sensitivity of MALDI-TOF-MS decreases as the size of the analyte increases, it is desirable to design PCR assays that yield products containing 200 or fewer base pairs.
To illustrate the applicability of mass spectrometry for PCR product detection, an assay was devised for amplifying a 75-bp product corresponding to bases 1626 to 1701 of the cystic fibrosis transmembrane conductance regulator gene. The most common mutation of this gene is the delta F508, which is a three-base deletion that produces a 72bp PCR product. Following PCR and a simple cleanup procedure, the product was detected by MALDI-TOF-MS. As a second example, PCR products were prepared from the genomic DNA of bacteria of the genus Legionella using primers that are available in a commercial kit. The resulting products--a 1 08-mer common to all members of the genus, and a 1 68-mer specific to L. pneumophila, the organism that causes Legionnaire's disease--were detected by ALDI-TOF-MS. Current challenges for MALDI-TOF analysis of PCR products include improved methods for removing salts and other reagents, and improvements in resolution and upper mass limit.
Sensitivity is also an active area of research for MALDI-TOF. The majority of a typical sample (1 µL of a 1 µM solution of DNA or protein) can be recovered following the MALDI measurement, suggesting that the analysis can be performed with much smaller sample sizes. We are currently developing sample preparation techniques using nanoliter and smaller volumes.
*Research supported by the Office of Health and Environmental Research, U.S. Department of Energy, and the Oak Ridge National Laboratory Director's Research and Development Program, under contract DE-AC05-840R21400 with Lockheed Martin Energy Systems, Inc.
[1] Health Sciences Research Division, ORNL
[2] Biology Division, ORNL
The submitted manuscript has been authorized by a contractor of the U.S. Government under contract No. DE-AC05-84OR21400. Accordingly, the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes.