2.50
Hdl Handle:
http://hdl.handle.net/10146/77968
Title:
Accelerated (32)P-HPLC for bulky DNA adducts.
Authors:
Nagy, Eszter; Cornelius, Michael G.; Moller, Lennart
Abstract:
Many compounds can react with DNA forming covalent modifications, so-called DNA adducts, which can influence crucial biological processes. DNA adducts from various DNA-damaging agents can act both as biomarkers and as a measurement of the actual damage of the genome. Therefore, they are of value in determining exposed or sensitive individuals or populations and in identifying agents that can induce DNA damage. A common method to measure DNA adducts is through DNA hydrolysis, adduct enrichment, (32)P-post-labelling and chromatographic separation. High-performance liquid chromatography (HPLC) with online radioactivity detection, the (32)P-HPLC (direct injection of the (32)P-labelled mixture into the HPLC with online (32)P-detection) method, gives both high sensitivity and good resolution of complex mixtures of DNA adducts. One limitation with this method is the capacity when dealing with large numbers of samples. The aim of this study was therefore to increase the analytical capacity by reducing analysis time of the (32)P-HPLC method. A change of HPLC columns to low backpressure columns and adaptation of elution conditions enabled a reduction in time per analysis from 100 to 20 min. This did not affect sensitivity but lowered chromatographic resolution, although bulky DNA adducts were still well resolved from other DNA components. This is useful when the total amount of DNA adducts is the primary interest. When high resolution is required, this can be achieved by gradient modifications, which increase the time required per analysis to 30 min. The accelerated (32)P-HPLC increases the capacity in number of samples 3- to 5-fold, depending on resolution requirements, without any negative effect on sensitivity in both in vitro and in vivo samples.
Citation:
Mutagenesis 2009, 24 (2):183-189
Journal:
Mutagenesis
Issue Date:
Mar-2009
URI:
http://hdl.handle.net/10146/77968
DOI:
10.1093/mutage/gen070
PubMed ID:
19098133
Additional Links:
http://mutage.oxfordjournals.org/cgi/content/full/24/2/183
Type:
Article
Language:
en
ISSN:
1464-3804
Sponsors:
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).
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorNagy, Eszter-
dc.contributor.authorCornelius, Michael G.-
dc.contributor.authorMoller, Lennart-
dc.date.accessioned2009-08-20T11:46:06Z-
dc.date.available2009-08-20T11:46:06Z-
dc.date.issued2009-03-
dc.identifier.citationMutagenesis 2009, 24 (2):183-189en
dc.identifier.issn1464-3804-
dc.identifier.pmid19098133-
dc.identifier.doi10.1093/mutage/gen070-
dc.identifier.urihttp://hdl.handle.net/10146/77968-
dc.description.abstractMany compounds can react with DNA forming covalent modifications, so-called DNA adducts, which can influence crucial biological processes. DNA adducts from various DNA-damaging agents can act both as biomarkers and as a measurement of the actual damage of the genome. Therefore, they are of value in determining exposed or sensitive individuals or populations and in identifying agents that can induce DNA damage. A common method to measure DNA adducts is through DNA hydrolysis, adduct enrichment, (32)P-post-labelling and chromatographic separation. High-performance liquid chromatography (HPLC) with online radioactivity detection, the (32)P-HPLC (direct injection of the (32)P-labelled mixture into the HPLC with online (32)P-detection) method, gives both high sensitivity and good resolution of complex mixtures of DNA adducts. One limitation with this method is the capacity when dealing with large numbers of samples. The aim of this study was therefore to increase the analytical capacity by reducing analysis time of the (32)P-HPLC method. A change of HPLC columns to low backpressure columns and adaptation of elution conditions enabled a reduction in time per analysis from 100 to 20 min. This did not affect sensitivity but lowered chromatographic resolution, although bulky DNA adducts were still well resolved from other DNA components. This is useful when the total amount of DNA adducts is the primary interest. When high resolution is required, this can be achieved by gradient modifications, which increase the time required per analysis to 30 min. The accelerated (32)P-HPLC increases the capacity in number of samples 3- to 5-fold, depending on resolution requirements, without any negative effect on sensitivity in both in vitro and in vivo samples.en
dc.description.sponsorshipECNIS (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).en
dc.language.isoenen
dc.relation.urlhttp://mutage.oxfordjournals.org/cgi/content/full/24/2/183en
dc.subjectBenz(a)Anthracenesen
dc.subjectCell Lineen
dc.subjectDNA Adductsen
dc.subjectPhosphorus Radioisotopesen
dc.subjectReference Standardsen
dc.subjectChromatographyen
dc.subject.meshBenz(a)Anthracenes-
dc.subject.meshCell Line-
dc.subject.meshChromatography, High Pressure Liquid-
dc.subject.meshDNA Adducts-
dc.subject.meshHumans-
dc.subject.meshPhosphorus Radioisotopes-
dc.subject.meshReference Standards-
dc.titleAccelerated (32)P-HPLC for bulky DNA adducts.en
dc.typeArticleen
dc.identifier.journalMutagenesisen
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