Abstract
The potential of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry for the characterization of polymerase chain reaction (PCR) amplified nucleic acids was evaluated. For that purpose, a "SNP toolbox" was constructed by cloning and PCR-mediated site-directed in vitro mutagenesis at nucleotide position (ntp) 16,519 of a sequence-verified fragment of the human mitochondrial genome (ntps 15,900-599). Confirmatory sequencing demonstrated that within the sequences of the clones one and the same base was mutated to all other bases. Using these clones or equimolar mixtures of these clones as PCR templates, 51-401-bp-long amplicons were generated, which were used to determine the upper size limits of PCR products for the unequivocal detection of sequence variations in homo- and heterozygous samples. Based on the high mass spectrometric performance of the applied time-of-flight mass spectrometer, the unequivocal genotyping of all kinds of single base exchanges in PCR amplicons from heterozygous samples with lengths up to 254 base pairs (bp) was demonstrated. Considering homozygous samples, the successful genotyping of single base substitutions in up to 401-bp-long PCR products was possible. Consequently, the described hyphenated technique represents one of the most powerful mass spectrometric genotyping assays available today.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.