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Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach

Moore, SA and Xeniou, O and Zeng, ZT and Humphreys, E and Burr, S and Gottschalg, E and Bingham, SA and Shuker, DEG (2010) Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach. ANALYTICAL BIOCHEMISTRY, 403 (1-2). pp. 67-73. ISSN 0003-2697

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Abstract

Immunoslot blot assays have been used for the analysis of many DNA adducts, but problems are frequently encountered in achieving reproducible results. Each step of the assay was examined systematically, and it was found that the major problems are in the DNA fragmentation step and the use of the manifold apparatus. Optimization was performed on both the malondialdehyde–deoxyguanosine (M1dG) adduct and the O6-carboxymethyl–deoxyguanosine (O6CMdG) adduct to demonstrate the applicability to other DNA adducts. Blood samples from the European Prospective Investigation on Cancer(EPIC) study (n = 162) were analyzed for M1dG adducts, and the data showed no correlation with adduct levels in other tissues, indicating that the EPIC blood samples were not useful for studying M1dG adducts. Blood samples from a processed meat versus vegetarian diet intervention (n = 6) were analyzed for O6CMdG, and many were below the limit of detection. The reduction of background adduct levels in standard DNA was investigated using chemical and whole genome amplification approaches. The latter gave a sensitivity improvement of 2.6 adducts per 107 nucleotides for the analysis of O6CMdG. Subsequentreanalysis for O6CMdG showed a weakly significant increase in O6CMdG on the processed meat diet compared with the vegetarian diet, demonstrating that further studies are warranted. 2010 Elsevier Inc. All rights reserved.

Item Type: Article
Uncontrolled Keywords: 0301 Analytical Chemistry, 0601 Biochemistry And Cell Biology, 0399 Other Chemical Sciences
Subjects: Q Science > QD Chemistry
Divisions: Pharmacy & Biomolecular Sciences
Publisher: Elsevier Masson
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Date Deposited: 22 May 2015 10:47
Last Modified: 07 Sep 2017 00:21
DOI or Identification number: 10.1016/j.ab.2010.04.015
URI: http://researchonline.ljmu.ac.uk/id/eprint/1154

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