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

McLuckie KI, Lamb JH, Sandhu JK, Pearson HL, Brown K, Farmer PB, Jones DJ - Nucleic Acids Res. (2006)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biomarkers and Prevention Group, The Biocentre, University of Leicester, University Road, Leicester, LE1 7RH, UK. kiem1@le.ac.uk

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.

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Outline of MALDI-MS site-selective mutation assay (SSMA-MS).
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fig1: Outline of MALDI-MS site-selective mutation assay (SSMA-MS).

Mentions: In order to address these limitations we describe in this manuscript a novel site-specific mutation assay which is an adaptation of the supF assay routinely used in our laboratory and benefits from the use of MALDI-ToF mass spectrometry (MALDI-MS) for the characterization of mutations. Synthetic deoxyoligonucleotides containing an O6-methyl-deoxyguanosine (O6-MedG) were inserted into the supF gene of the pSP189 plasmid (19,20). When the plasmid is transfected into and recovered from, human cells the presence of the synthetic insert acts as a frameshift mutation, thus inactivating the supF gene, resulting in the production of white mutant colonies (Figure 1). PCR followed by digestion with Mbo11/Mnl1 yields small (12 base) double stranded deoxyoligonucleotides which were then analysed by MALDI-MS (Figure 2). The increased throughput and reduced cost of using mass spectrometry for analysis will allow the rapid screening of the mutations induced by multiple DNA lesions produced by a single compound in different sequence contexts. This technique can ultimately provide insight into the initiation processes of carcinogenesis.


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

McLuckie KI, Lamb JH, Sandhu JK, Pearson HL, Brown K, Farmer PB, Jones DJ - Nucleic Acids Res. (2006)

Outline of MALDI-MS site-selective mutation assay (SSMA-MS).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1761429&req=5

fig1: Outline of MALDI-MS site-selective mutation assay (SSMA-MS).
Mentions: In order to address these limitations we describe in this manuscript a novel site-specific mutation assay which is an adaptation of the supF assay routinely used in our laboratory and benefits from the use of MALDI-ToF mass spectrometry (MALDI-MS) for the characterization of mutations. Synthetic deoxyoligonucleotides containing an O6-methyl-deoxyguanosine (O6-MedG) were inserted into the supF gene of the pSP189 plasmid (19,20). When the plasmid is transfected into and recovered from, human cells the presence of the synthetic insert acts as a frameshift mutation, thus inactivating the supF gene, resulting in the production of white mutant colonies (Figure 1). PCR followed by digestion with Mbo11/Mnl1 yields small (12 base) double stranded deoxyoligonucleotides which were then analysed by MALDI-MS (Figure 2). The increased throughput and reduced cost of using mass spectrometry for analysis will allow the rapid screening of the mutations induced by multiple DNA lesions produced by a single compound in different sequence contexts. This technique can ultimately provide insight into the initiation processes of carcinogenesis.

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biomarkers and Prevention Group, The Biocentre, University of Leicester, University Road, Leicester, LE1 7RH, UK. kiem1@le.ac.uk

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.

Show MeSH
Related in: MedlinePlus