A mass spectrometry based hybridisation assay for single nucleotide polymorphism analysis
A mass spectrometry based hybridisation assay for single nucleotide polymorphism analysis
Peptide nucleic acid is an analogue of deoxyribonucleic acid in which the phosphate backbone has been replaced with an achiral, uncharged polyamide backbone. This modification allows peptide nucleic acid to form duplexes with high affinity and sequence specificity with complementary deoxyribonucleic acid strands.
An approach that uses peptide nucleic acid probes in a hybridisation assay to detect single nucleotide polymorphisms has been developed. The probes hybridise to an immobilised single stranded polymerase chain reaction product and the peptide nucleic acid/deoxyribonucleic acid/bead conjugate is analysed directly by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. The genotype of the deoxyribonucleic acid is determined by the molecular mass of the peptide nucleic acid probe released. From one peptide nucleic acid scaffold four different types of peptide nucleic acid probes have been synthesised; an unmodified probe, a charge tagged probe, a probe with charge tagged mass marker and a probe with isotopically labelled charge tagged photo-cleavable mass marker.
Cystic fibrosis mutation analysis was chosen to evaluate the peptide nucleic acid hybridisation assay; in particular, studies have focused on mutations on the W1282X locus. The peptide nucleic acid probes have shown discrimination against mismatches and the neutral backbone does not form cation adducts like conventional deoxyribonucleic acid probes, which can hamper the sensitivity and resolution of detection by mass spectrometry. All four types of peptide nucleic acid probe have been successfully used in the hybridisation assay to give unambiguous detection of point mutations.
University of Southampton
Ball, Rachel Jennifer
31017fec-283f-4c96-a822-fcbd4765ef2e
2005
Ball, Rachel Jennifer
31017fec-283f-4c96-a822-fcbd4765ef2e
Ball, Rachel Jennifer
(2005)
A mass spectrometry based hybridisation assay for single nucleotide polymorphism analysis.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Peptide nucleic acid is an analogue of deoxyribonucleic acid in which the phosphate backbone has been replaced with an achiral, uncharged polyamide backbone. This modification allows peptide nucleic acid to form duplexes with high affinity and sequence specificity with complementary deoxyribonucleic acid strands.
An approach that uses peptide nucleic acid probes in a hybridisation assay to detect single nucleotide polymorphisms has been developed. The probes hybridise to an immobilised single stranded polymerase chain reaction product and the peptide nucleic acid/deoxyribonucleic acid/bead conjugate is analysed directly by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. The genotype of the deoxyribonucleic acid is determined by the molecular mass of the peptide nucleic acid probe released. From one peptide nucleic acid scaffold four different types of peptide nucleic acid probes have been synthesised; an unmodified probe, a charge tagged probe, a probe with charge tagged mass marker and a probe with isotopically labelled charge tagged photo-cleavable mass marker.
Cystic fibrosis mutation analysis was chosen to evaluate the peptide nucleic acid hybridisation assay; in particular, studies have focused on mutations on the W1282X locus. The peptide nucleic acid probes have shown discrimination against mismatches and the neutral backbone does not form cation adducts like conventional deoxyribonucleic acid probes, which can hamper the sensitivity and resolution of detection by mass spectrometry. All four types of peptide nucleic acid probe have been successfully used in the hybridisation assay to give unambiguous detection of point mutations.
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Published date: 2005
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Local EPrints ID: 465502
URI: http://eprints.soton.ac.uk/id/eprint/465502
PURE UUID: ee9b5e2b-1e19-4c9d-bf2b-50492eddd339
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Date deposited: 05 Jul 2022 01:29
Last modified: 16 Mar 2024 20:13
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Author:
Rachel Jennifer Ball
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