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Additional resources for Methods in Virology, Volume 8
310. 1. HYBRIDIZATION OF VIRAL NUCLFIC ACIDS 25 eral nucleotides away from the axis on each strand. This staggered cut yields DNA fragments with cohesive termini that can readily recombine with other such termini to form recombinant molecules. The optimal reaction conditions for each particular enzyme are indicated on the manufacturer's specification sheet. To avoid possible errors in buffer conditions it is generally advisable to prepare a 10 x stock solution of the appropriate buffer that can be stored at - 2 0 ° C indefinitely.
Electrophoresis in an alkaline gel will disclose only the individual DNA species a and e. Exonuclease VII will di gest only the single-stranded tails, not any internal loops, and will identify the length of the DNA fragment between the 5' and 3 ' ends of the RNA. The reaction is set up by precipitating the appropriate amount of DNA and RNA with ethanol. To detect RNAs of very low abundance, the con centration of RNA can be increased up to 500 μg in a 100-μ1 hybridization reaction mixture. 5).
Originally developed as a preparatory method, fingerprinting 41 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-470208-2 42 O. M. KEW, E. K. NOTTAY, AND J. F. OBUESKI is currently most frequently applied as an analytical method for comparing the genomes of RNA viruses. Comparisons are based on the principle that the large, structurally unique oligonucleotides separate into patterns, or "fingerprints," that are highly characteristic of the RNA sequence from which they derive.