JBC, Vol. 253, Issue 23, 8400-8405, Dec, 1978
A hybridization procedure for the isolation of specific RNA segments applied to the analysis of bacteriophage Qbeta RNA
A. Shapira and M. A. Billeter
A method for the isolation of RNA fragments originating from defined
regions of bacteriophage Qbeta RNA minus strands is described. Large RNase
T1 oligonucleotides were isolated on a preparative scale from Qbeta RNA.
The nucleotide sequences (13 to 26 nucleotides) and map positions of these
oligonucleotides were known from previous work (Billeter, M. A. (1978) J.
Biol. Chem. 253, 8381-8389). After addition of AMP residues (50 in the
average) using terminal adenylate transferase, these pure oligonucleotides
were hybridized to 32P-labeled Qbeta RNA minus strands synthesized in
vitro. Fragments in the size range of 100 to 500 nucleotides were then
generated by partial digestion with RNase T1. Fragments hybridized to such
oligonucleotides were recovered by chromatography on poly(U)-Sephadex and
then resolved according to their size by polyacrylamide gel
electrophoresis. The specificity and reproducibility of the method as well
as its suitability for the sequence analysis of Qbeta RNA was verified by
using in particular a linker oligonucleotide derived from a Qbeta RNA
region near the 3' end. The sequence catalogues of the RNase T1 and RNase A
oligonucleotides of two fragments isolated in this way, 202 and 310
nucleotides in length, were established and all fragments isolated were
shown to contain a sequence complementary to the linker oligonucleotide.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
