Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations.
The random amplified polymorphic DNA (RAPD) assay and related techniques like the arbitrarily primed polymerase chain reaction (AP-PCR) have been shown to detect genotoxin-induced DNA damage and mutations. The changes occurring in RAPD profiles following genotoxic treatments include variation in band intensity as well as gain or loss of bands. However, the interpretation of the molecular events responsible for differences in the RAPD patterns is not an easy task since different DNA alterations can induce similar type of changes. In this study, we evaluated the effects of a number of DNA alterations on the RAPD profiles. Genomic DNA from different species was digested with restriction enzymes, ultrasonicated, treated with benzo[a]pyrene (B[a]P) diol epoxide (BPDE) and the resulting RAPD profiles were evaluated. In comparison to the enzymatic DNA digestions, sonication caused greater changes in the RAPD patterns and induced a dose-related disappearance of the high molecular weight amplicons. A DNA sample substantially modified with BPDE caused very similar changes but amplicons of low molecular weight were also affected. Appearance of new bands and increase in band intensity were also evident in the RAPD profiles generated by the BPDE-modified DNA. Random mutations occurring in mismatch repair-deficient strains did not cause any changes in the banding patterns whereas a single base change in 10-mer primers produced substantial differences. Finally, further research is required to better understand the potential and limitations of the RAPD assay for the detection of DNA damage and mutations.
Atienzar
Franck A
FA
School of Biological Sciences, Plymouth Environmental Research Centre, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, UK. fatienzar@skynet.be
Venier
Paola
P
Jha
Awadhesh N
AN
Depledge
Michael H
MH
D020137:Base Pair Mismatch; D020029:Base Pairing; D001580:Benzopyrenes / Q000633:toxicity; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics*; D053938:DNA Fragmentation / Q000187:drug effects; D004252:DNA Mutational Analysis / Q000379:methods*; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism
D001580:Benzopyrenes; D018736:DNA Adducts; C072019:benzo(a)pyrene diolepoxide I; D004247:DNA; C090528:DNA modification methylase HindIII; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015246:Deoxyribonuclease EcoRI