The early melting of closed duplex DNA: analysis by banding in buoyant neutral rubidium trichloroacetate.
The early melting of closed duplex DNA: analysis by banding in buoyant neutral rubidium trichloroacetate.
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闭合双链体 DNA 的早期熔化：通过浮力中性三氯乙酸铷中的条带进行分析。
DOI：
10.1093/nar/8.5.1145
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发表时间：
1980
影响因子：
14.9
通讯作者：
Bauer,WR
中科院分区：
文献类型：
--
作者：
Burke,RL;Bauer,WR
水性RbTCA允许在室温和中性pH下天然和变性DNA的浮力条带。这种溶剂的独特性质是封闭的圆形，下缠绕的DNA（I）从相应的切口（II）物种的浮力分辨率。本文报道了PM-2 DNA I与天然PM-2 DNA II物理分离的条件，在25°C下，在3.3 H RbTCA中的浮力分离为1.27 ma/ml。切口DNA和闭合DNA之间的分离随着温度升高而增加，直至35.5°C，在该温度下PM-2 DNA II协同熔化并随后沉淀。封闭DNA的等温浮力密度随着封闭DNA的连接数（Lk）的减小而线性增加。封闭DNA的早期解链可以通过RbTCA中的浮力条带以高精度监测，可以检测PM-2 DNA中少至40个碱基对（10，000个碱基对）的破坏。封闭DNA中的连接数保守的限制要求双链体windina的变化伴随着suoer-coilina的补偿性变化。我们估计PM-2 DNA I的连接数不足为0.094转/decibase对。这一结果允许估计EtdBr的解旋角，θ，通过比较与其他确定的连接数不足，这取决于值θ。这里得到的结果是，θ = 27.7° ± 0.5°，并且在15° − 35°范围内与温度近似无关。
Aqueous RbTCA permits the buoyant banding of both native and denatured DNA at room temperature and neutral pH. A unique property of this solvent is the buoyant resolution of closed circular, underwound DNA (I) from the corresponded nicked (II) species. Conditions are reported here in which PM-2 DNA I is physically resolved from native PM-2 DNA II, the buoyant separation being 1.27 ma/ml in 3.3 H RbTCA at 25°C. The separation between nicked and closed DNAs increases with temperature up to 35.5°C, at which PM-2 DNA II cooperatively melts and subsequently pellets. The isothermal buoyant density of a closed DNA increases linearly as the linking number (Lk) of the closed DNA decreases. The early melting of closed DNA may be monitored with high precision by buoyant banding in RbTCA, it being possible to detect the disruption of as few as 40 base pairs in PM-2 DNA (10,000 base pairs). The constraint that the linking number be conserved in closed DNA requires that a change in duplex windina be accompanied by a compensating change in suoer-coilina. We estimate the linking number deficiency of PM-2 DNA I to be 0.094 turns per decibase pair. This result permits the estimation of the EtdBr unwinding angle, ø, by comparison with alternative determinations of the linking number deficiency which depend upon the value of ø. The result obtained here is that ø = 27.7° ± 0.5° and is approximately independent of temperature over the range 15° − 35°.