Use of thermostable and Escherichia coli RNase H in RNA mapping studies.
A recently introduced thermostable RNase H was tested to determine its effectiveness in RNase H mapping reactions. Procedures are described which should have general use with both the thermostable and the Escherichia coli RNase H enzymes. Using the thermostable RNase H at higher temperatures extends the range of oligodeoxyribonucleotide/RNA combinations that yield satisfactory results. Northern blot analyses of total RNA was used to demonstrate that native RNAs can be analyzed by oligodeoxyribonucleotide directed RNase H digestion with minimal sample processing as long as care is taken to maintain thermal stringency both during reaction assembly and termination. Increased thermal stringency allows for higher DNA concentrations to ensure complete site-specific digestion of target RNAs or to permit simultaneous cleavage with multiple oligodeoxyribonucleotides. Partial digests can also be controlled by manipulating oligodeoxyribonucleotide concentrations. In addition, the thermostable RNase H was shown to be active at magnesium ion concentrations as low as 0.1 mM. This allows for optimization of Mg2+ effects on overall sample integrity and DNA/RNA interactions over at least a 20-fold range (2.0-0.1 mM).
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Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins 80523-1870, USA.
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification*; D051381:Rats; D016914:Ribonuclease H; D013552:Swine; D013696:Temperature
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D016914:Ribonuclease H