Genotyping short tandem repeats by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry.

摘要

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) offers great promise as a method to genotype individuals by direct mass measurement of intact polymerase chain reaction (PCR) amplicons. Particularly attractive types of polymorphisms for which ESI mass spectrometry would improve upon existing techniques, are short tandem repeat (STR) sequences also known as microsatellites. The detection of oligonucleotides and PCR amplicons of STR sequences by mass spectrometry presents significant challenges. To detect the small volume and low concentrations produced during the PCR, implementation of a durable nanoelectrospray format was accomplished. A PCR purification methodology based on ethanol precipitation, microdialysis and organic modifiers is used that effectively reduces cation adduction to DNA in the gas phase. The purification and nanoelectrospray strategy allows the routine attainment of quality mass spectra of PCR amplicons leading to accurate genotyping of a STR locus. A dual electrospray source is developed for internal calibration improving mass accuracy and allowing the future genotyping of compound STR loci. To illustrate the potential of STR genotyping by ESI-MS for clinical situations, initial work for high throughput relying on flow injection analysis is accomplished for two PCR amplicons. Finally, the gas-phase sequencing of oligonucleotides based on known trinucleotide and tetranucleotide repeats is examined with the incorporation of nucleotide analogs to alter preferential pathways during low energy fragmentation for the future intent of identifying interruptions in within STR sequences.