A procedure for cloning genomic DNA fragments with increasing thermoresistance.
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.
Dipartimento di Biologia Cellulare, Università della Calabria, 87030, Arcavacata di Rende (CS), Italy.
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Bellizzi
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Cartolano
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Annesi
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Tomaino
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Sgaramella
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Losso
D010582:Bacteriophage lambda; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry; D015723:Gene Library; D015894:Genome, Human; D006358:Hot Temperature; D006801:Humans; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry; D015183:Restriction Mapping; D013816:Thermodynamics
D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D004247:DNA