Nucleic Acids Research Advance Access originally published online on January 26, 2007
Nucleic Acids Research 2007 35(4):e28; doi:10.1093/nar/gkl1123
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Nucleic Acids Research, 2007, Vol. 35, No. 4 e28
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS
1Trillion Genomics Ltd, Babraham Research Campus, Babraham, Cambridge CB2 4AT, UK, 2Biosciences Building, Crown Street, School of Biological Sciences, University of Liverpool, Liverpool L69 3BX, and 3School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK
*To whom correspondence should be addressed. Tel: + 44(0) 1223 362541; Fax: + 44(0) 8700 940151; E-mail: andrewthompson{at}trilliongenomics.com
Received November 3, 2006. Revised December 7, 2006. Accepted December 8, 2006.
The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.