Development of a novel site-specific mutagenesis assay using MALDI-ToF MS (SSMA-MS).
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.
McLuckie
Keith I E
KI
Cancer Biomarkers and Prevention Group, The Biocentre, University of Leicester, University Road, Leicester, LE1 7RH, UK. kiem1@le.ac.uk
Lamb
John H
JH
Sandhu
Jatinderpal K
JK
Pearson
Helen L
HL
Brown
Karen
K
Farmer
Peter B
PB
Jones
Donald J L
DJ
D002460:Cell Line; D018736:DNA Adducts / Q000032:analysis; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000737:chemistry / Q000592:standards; D010957:Plasmids / Q000235:genetics; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000592:standards; D014162:Transfection
D018736:DNA Adducts; D009838:Oligodeoxyribonucleotides; C015558:O(6)-methyl-2'-deoxyguanosine; D003849:Deoxyguanosine