Hempel K, Mildenberger E
Int J Radiat Biol Relat Stud Phys Chem Med. 1987 Jul;52(1):125-38. doi: 10.1080/09553008714551551.
Covalently closed circular double-stranded DNA (CC) of native plasmids was used to determine the yield of single strand breaks (ssb) and double strand breaks (dsb) as a consequence of X-irradiation. One ssb transforms DNA of the CC form to the nicked circular form (NC), whereas one dsb produced either directly or from random coincidence of single strand breaks transforms DNA of the CC as well as of the NC form to linear DNA molecules (LI form). Plasmids with more than one dsb are cleaved to linear fragments. DNA (30-800 micrograms/ml) was irradiated in air-saturated sodium phosphate buffer. The different forms of DNA were separated by gel electrophoresis and their amounts measured fluorometrically using ethidium bromide. Large linear DNA fragments with the same electrophoretic mobility as the LI form were considered by using a curve-fitting procedure. From the quantitative changes of each conformation D37 values of ssb and dsb were calculated as a function of the DNA concentration. Finally G-values were calculated by competition plots. The following yields were determined: Gssb 3.4 X 10E-8 molJ-1, and Gdsb 3.3 X 10E-10 molJ-1. Gdsb refers only to those dsb produced directly. Yields are related to strand breaks without further treatment by heat or alkali.
天然质粒的共价闭合环状双链DNA（CC）被用于测定X射线照射后单链断裂（ssb）和双链断裂（dsb）的产率。一个单链断裂会将CC形式的DNA转化为带切口的环状形式（NC），而一个双链断裂，无论是直接产生的还是由单链断裂的随机巧合产生的，都会将CC形式以及NC形式的DNA转化为线性DNA分子（LI形式）。具有多个双链断裂的质粒会被切割成线性片段。将DNA（30 - 800微克/毫升）在空气饱和的磷酸钠缓冲液中进行照射。通过凝胶电泳分离不同形式的DNA，并使用溴化乙锭通过荧光法测量它们的量。通过曲线拟合程序来考虑与LI形式具有相同电泳迁移率的大线性DNA片段。根据每种构象的定量变化，计算单链断裂和双链断裂的D37值作为DNA浓度的函数。最后通过竞争图计算G值。测定了以下产率：Gssb为3.4×10⁻⁸摩尔/焦耳，Gdsb为3.3×10⁻¹⁰摩尔/焦耳。Gdsb仅指直接产生的那些双链断裂。产率与未经加热或碱进一步处理的链断裂有关。
