Development of a novel site-specific mutagenesis assay using MALDI-ToF MS (SSMA-MS).

2.50
Hdl Handle:
http://hdl.handle.net/10146/25453
Title:
Development of a novel site-specific mutagenesis assay using MALDI-ToF MS (SSMA-MS).
Authors:
McLuckie, Keith I.E.; Lamb, John H.; Sandhu, Jatinderpal K.; Pearson, Helen L.; Brown, Karen; Farmer, Peter B.; Jones, Donald J.L.
Abstract:
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 O(6)-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.
Citation:
Nucleic Acids Res. 2006, 34 (22):e150
Journal:
Nucleic Acids Research
Issue Date:
2006
URI:
http://hdl.handle.net/10146/25453
DOI:
10.1093/nar/gkl745
PubMed ID:
17130165
Additional Links:
http://nar.oxfordjournals.org/cgi/content/full/34/22/e150; http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17130165
Type:
Article
Language:
en
ISSN:
1362-4962
Sponsors:
This work was supported by the Medical Research Council, UK (G0100873). The authors of this paper are partners of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No 513943). Funding to pay the Open Access publication charges for this article was provided by the MRC.
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorMcLuckie, Keith I.E.-
dc.contributor.authorLamb, John H.-
dc.contributor.authorSandhu, Jatinderpal K.-
dc.contributor.authorPearson, Helen L.-
dc.contributor.authorBrown, Karen-
dc.contributor.authorFarmer, Peter B.-
dc.contributor.authorJones, Donald J.L.-
dc.date.accessioned2008-05-12T09:34:59Z-
dc.date.available2008-05-12T09:34:59Z-
dc.date.issued2006-
dc.identifier.citationNucleic Acids Res. 2006, 34 (22):e150en
dc.identifier.issn1362-4962-
dc.identifier.pmid17130165-
dc.identifier.doi10.1093/nar/gkl745-
dc.identifier.urihttp://hdl.handle.net/10146/25453-
dc.description.abstractWe 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 O(6)-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.en
dc.description.sponsorshipThis work was supported by the Medical Research Council, UK (G0100873). The authors of this paper are partners of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No 513943). Funding to pay the Open Access publication charges for this article was provided by the MRC.en
dc.language.isoenen
dc.relation.urlhttp://nar.oxfordjournals.org/cgi/content/full/34/22/e150en
dc.relation.urlhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17130165en
dc.subject.meshCell Line-
dc.subject.meshDNA Adducts-
dc.subject.meshDeoxyguanosine-
dc.subject.meshEscherichia coli-
dc.subject.meshGenetic Vectors-
dc.subject.meshHumans-
dc.subject.meshMutagenesis, Site-Directed-
dc.subject.meshOligodeoxyribonucleotides-
dc.subject.meshPlasmids-
dc.subject.meshReference Standards-
dc.subject.meshSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization-
dc.subject.meshTransfection-
dc.titleDevelopment of a novel site-specific mutagenesis assay using MALDI-ToF MS (SSMA-MS).en
dc.typeArticleen
dc.identifier.journalNucleic Acids Researchen

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