Bellizzi D, Cartolano A S, Annesi F, Tomaino C, Sgaramella V, Losso M A
Dipartimento di Biologia Cellulare, Università della Calabria, 87030, Arcavacata di Rende (CS), Italy.
Gene. 1998 Sep 28;219(1-2):63-71. doi: 10.1016/s0378-1119(98)00377-1.
Genomic DNAs have been cleaved by restriction or sonication, and the resulting double-stranded fragments have been exposed to increasing temperatures. This treatment may induce the helix-coil transition either in a single or in several steps, depending on the size and composition of the duplexes. Eventually, a critical temperature is reached at which each duplex melts completely and the two constitutive single strands separate. A transition interval can thus be defined for each duplex by the temperature at which the earliest strand separation takes place and that at which the most resistant double-stranded core collapses. If solutions containing a mixture of DNA duplexes are exposed to temperatures within their transition intervals, three kinds of molecules should originate: (1) duplexes that have not yet initiated the melting phase; (2) duplexes that have undergone only partial melting; and (3) single strands that derive from fully melted duplexes. If the heated solutions are quickly cooled to 0 degreesC, only the molecules from the first two classes can be ligated to a compatibly ended vector and cloned: class (1) are intact duplexes, and class (2) are molecules that snap immediately back to fully duplex structures: both are double-stranded. Conversely, the single strands of class (3) may not reanneal and thus be neither ligated nor cloned. We have tested the procedure on restricted coliphage lambda DNA, in view of its compartmentalized organization and known sequence. Then, we have applied it to human genomic DNA fragmented by sonication. After cloning of the available duplexes in a bacterial plasmid, libraries of molecules endowed with a progressively higher thermoresistance can be prepared for thermodynamic and genomic studies.
基因组DNA已通过限制性内切酶切割或超声处理进行裂解，产生的双链片段被置于不断升高的温度下。这种处理可能会以单步或多步诱导螺旋-卷曲转变，这取决于双链体的大小和组成。最终，会达到一个临界温度，此时每个双链体完全解链，两条组成单链分离。因此，可以通过最早发生链分离的温度和最稳定的双链核心解链的温度为每个双链体定义一个转变区间。如果将含有DNA双链体混合物的溶液置于其转变区间内的温度下，应该会产生三种分子：（1）尚未开始解链阶段的双链体；（2）仅经历部分解链的双链体；（3）源自完全解链双链体的单链。如果将加热后的溶液迅速冷却至0℃，只有前两类分子可以与具有兼容末端的载体连接并克隆：第（1）类是完整的双链体，第（2）类是立即恢复为完全双链结构的分子：两者都是双链的。相反，第（3）类单链可能不会重新退火，因此既不能连接也不能克隆。鉴于其分区组织和已知序列，我们已在限制性噬菌体λDNA上测试了该程序。然后，我们将其应用于通过超声处理破碎的人类基因组DNA。在将可用的双链体克隆到细菌质粒中后，可以制备具有逐渐更高耐热性的分子文库，用于热力学和基因组研究。
