Philip Ross, Ph.D., Research Scientist
DNA Technology Development Branch, Center for Medical and Molecular Genetics, Armed Forces Inst. of Pathology, Rockville, MD

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Mass spectrometry, particularly MALDI-TOFMS, has received considerable attention as a valuable technology for high throughput screening of genetic polymorphisms. The technique has the potential to detect multiplexed analyses in an individual sample in a matter of seconds, and to scan through 100 samples in an hour. The main drawback is that MALDI-TOFMS requires nucleic acid samples to be extensively purified for optimum performance. For this reason, alternative chemistries must be designed to facilitate the purification process. We have developed a number of such strategies for various genotyping applications that are simple, automatable and compatible with MALDI-TOFMS.

Several approaches are based on direct sizing of DNA fragments from PCR or multiplex PCR reactions. These applications include microchip PCR product analysis, short tandem repeats, and direct size analysis for single nucleotide polymorphism screening. The sample handling method is based on solid phase immobilization, followed by washing and release of DNA strands in a simple, one tub procedure directly following PCR. The DNA sizing results from this approach demonstrate the unparalleled molecular weight accuracy and reproducibility of MALDI-TOFMS. For short tandem repeat analysis, sample preparation and typing can be accomplished in 15 minutes post-PCR, and the accuracy and stability of the MALDI-TOFMS instrumentation eliminates the need for molecular weight calibration. We have also optimized the chemistry and instrument performance such that it is readily possible to identify single base substitutions by direct molecular weight determination. Incorporation of microchip-based amplification technology with our MALDI-TOFMS approach permits a complete analysis to be performed in under an hour. Most recently, our mass spectrometric approaches have been expanded to include multiplex short tandem repeat analysis. At this point, research effort is more focused on PCR optimization for successful deployment of multiplex STR systems.

We have also developed a number of post-PCR genotyping approaches which make use of peptide nucleic acid (PNA) hybridization probes and enzymatic oligonucleotide extension. In both cases, mass spectrometry provides exceptional molecular weight accuracy to permit unambiguous determination of polymorphisms. PNAs have a promising role in the context of MALDI-TOFMS based genotyping, since hybridization protocols can be carried out rapidly and under varied buffer conditions. Results from application of a solid phase PNA probe assay for single base polymorphisms in mtDNA will be presented.

A final area of exploration is the development of oligonucleotide extension assays for MALDI-TOFMS typing of mtDNA. With oligonucleotide extension, we aim to sequence small regions of mtDNA to eventually obtain full coverage of hypervariable regions. After completion of the assay, a simple purification protocol to facilitate the rapid analysis capabilities of MALDI-TOFMS is required. An approach has been devised in our lab in which a capture reagent is used to directly treat extension reactions such that informative products can be washed and then analyzed by MALDI-TOFMS in a few minutes. These approaches are currently under investigation as automated and or array based technologies for high throughput scale-up.