Detection by Rapd of Genetic Alterations in Vitro: Amplification and Conservation Conditions of Dna Extracts

Abstract

The technique of random amplified polymorphic DNA (RAPD) permits the study of genetic ecotoxicology without having to face the problem posed by the high number of chromosomes generally presented by the fish species used in traditional methods. Additionally, the use of in vitro systems allows the study of a large number of samples in environmental-risk assessment. Good standardization of the parameters involved in the RAPD reaction, such as primer concentration and the DNA template used and its integrity, is fundamental for obtaining reliable and repeatable results. This is especially important when the differences in DNA fingerprints between control cells and cells exposed to genotoxic agents are interpreted as toxic-dependent alterations. The use of more than one primer increases sensitivity in the detection of such differences, provided the amplification is carried out under optimum conditions. This article demonstrates that the conditions established for certain primers in previous studies can be acceptable for others, independent of the complexity of band patterns generated. Furthermore, the integrity of the DNA is shown to be stable for several months in the genomic extracts stored at 4 degrees C, which to a large extent facilitates the application of this methodology.

The use of Fish Cells in Ecotoxicology: The Report and Recommendations of ECVAM Workshop 47,

“…Therefore, genotoxicity detected in a fish cell line exposed to environmental genotoxicants will most likely also be manifested in vivo, when fish are exposed to the same agent. It is probable that the agent could also alter the genomic complement of a given population (105,(107)(108)(109).…”

Risk Assessment of Food Coloring Agents on DNA Damage Using RAPD Markers

“…These effects may be correlated with structural rearrangements in DNA caused by different types of DNA damages [14]. The variation in band intensity and disappearance of some bands may correlate with level of photoproducts in DNA templates after treatment, which can reduce the number of binding sites for Taq polymerase.…”

Evidence for DNA-diquat interaction and cytotoxicity in in vitro rat cells

“…A number of concerns have been raised over the interaction between pesticides and DNA. The formation of DNA adducts was reported to be involved in mutagenic and carcinogenic processes and to lead to cell death (Becerril and Ferrero 2002).…”