e150.pdf (5.45 MB)
Download file

Development of A Novel Site-Specific Mutagenesis Assay Using MALDI-ToF MS (SSMA-MS).

Download (5.45 MB)
journal contribution
posted on 24.10.2007, 10:10 by Keith I.E. McLuckie, J.H. Lamb, J. Sandhu, H. Pearson, K. Brown, Peter B. Farmer, D.J. Jones
We have developed and validated a novel site-specific mutagenesis assay, termed SSMA-MS, which incorporates MALDI-ToF mass spectrometry (MALDI-MS) analysis as a means of determining the mutations induced by a single DNA adduct. The assay involves ligating an adducted deoxyoligonucleotide into supF containing pSP189 plasmid. The plasmid is transfected into human Ad293 kidney cells allowing replication and therefore repair or a mutagenic event to occur. Escherichia coli indicator bacteria are transformed with recovered plasmid and plasmids containing the insert are identified colormetrically, as they behave as frameshift mutations. The plasmid is then amplified and digested using a restriction cocktail of Mbo11 and Mnl1 to yield 12 bp deoxyoligonucleotides, which are characterized by MALDI-MS. MALDI-MS takes advantage of the difference in molecular weight between bases to identify any induced mutations. This analysis method therefore provides qualitative and quantitative information regarding the type and frequency of mutations induced. This assay was developed and validated using an O6-methyl-2′-deoxyguanosine adduct, which induced the expected GC→AT substitutions, when replicated in human or bacterial cells. This approach can be applied to the study of any DNA adduct in any biologically relevant gene sequence (e.g. p53) in human cells and would be particularly amenable to high-throughput analysis.

History

Citation

Nucleic Acids Research, 2006, 34 (22), p.e150

Published in

Nucleic Acids Research

Publisher

Oxford University Press (OUP)

issn

0305-1048

Available date

24/10/2007

Notes

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.;This paper was published as Nucleic Acids Research, 2006, 34 (22), p.e150. It is also available from http://nar.oxfordjournals.org/content/34/22/e150. DOI: 10.1093/nar/gkl745

Language

en

Usage metrics

Categories

Keywords

Exports