Genotyping complex short tandem repeats using electrospray ionization Fourier transform ion cyclotron resonance multi-stage mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is a rapidly emerging universal platform with the ability to provide detailed information regarding genetic variation and the up- and down regulation of their cognate gene products. Herein, we report our progress towards the development of ESI-FTICR mass spectrometry for the characterization of genomic regions which contain both a length and sequence polymorphism (i.e., complex short tandem repeats). Specifically, it is demonstrated for the first time that a high-quality ESI-FTICR mass spectrum of a 82-bp double-stranded PCR product derived from a single, 50 mu L PCR reaction with less than 10 x 10(-15) moles injected into the mass spectrometer can be routinely obtained. rt is important to note that each measurement, which translates to an accurate genotype, is completed on the timescale of seconds. Progress towards the implementation of now-injection analysis methodology to increase the throughput is also presented using an alternating injection of a 15-mer and 16-mer oligonucleotide. It is estimated that under our current configuration, which is very preliminary, about 250 genotypes could be generated per 24-hour time period. Furthermore, the gas-phase decomposition of large PCR products will clearly result in poor sensitivity due to the distribution of the molecular ion signal over numerous product-ions; many of which are not useful for detecting microheterogeneity within the repeating sequence (e.g., depurination). In an effort to channel the fragmentation of DNA sequences with a repeating motif into fewer product ions, 7-deaza adenosine was incorporated into a 16-mer oligonucleotide and dissociated using sustained off-resonance irradiation collision-induced dissociation. Comparison of the control sequence with the modified sequence clearly indicates that this is a highly effective strategy with current efforts directed at the dissociation of PCR products with 7-deaza punines incorporated.