Novel method for quantifying radiation-induced single-strand-break yields in plasmid DNA highlights 10-fold discrepancy.
Anal Biochem
; 417(2): 242-6, 2011 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-21741945
ABSTRACT
The widely used agarose gel electrophoresis method for assessing radiation-induced single-strand-break (SSB) yield in plasmid DNA involves measurement of the fraction of relaxed-circular (C) form that migrates independently from the intact supercoiled (SC) form. We rationalized that this method may underestimate the SSB yield since the position of the relaxed-circular form is not altered when the number of SSB per DNA molecule is >1. To overcome this limitation, we have developed a novel method that directly probes and quantifies SSBs. Supercoiled (3)H-pUC19 plasmid samples were irradiated with γ-rays, alkali-denatured, dephosphorylated, and kinated with γ-[(32)P]ATP, and the DNA-incorporated (32)P activities were used to quantify the SSB yields per DNA molecule, employing a standard curve generated using DNA molecules containing a known number of SSBs. The same irradiated samples were analyzed by agarose gel and SSB yields were determined by conventional methods. Comparison of the data demonstrated that the mean SSB yield per plasmid DNA molecule of [21.2±0.59]×10(-2)Gy(-1) as measured by direct probing is ~10-fold higher than that obtained from conventional gel-based methods. These findings imply that the SSB yields inferred from agarose gels need reevaluation, especially when they were utilized in the determination of radiation risk.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Circular
/
DNA Super-Helicoidal
/
Eletroforese em Gel de Ágar
/
Quebras de DNA de Cadeia Simples
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Estados Unidos