Nucleic Acids Research, Vol 25, Issue 9 1830-1835, Copyright © 1997 by Oxford University Press
R Fislage, M Berceanu, Y Humboldt, M Wendt and H Oberender
We have developed a primer set for a prokaryotic differential display of
mRNA in the Enterobacteriaceae group. Each combination of ten 10mer and ten
11mer primers generates up to 85 bands from total Escherichia coli RNA,
thus covering expressed sequences of a complete bacterial genome. Due to
the lack of polyadenylation in prokaryotic RNA the type T11VN anchored
oligonucleotides for the reverse transcriptase reaction had to be replaced
with respect to the original method described by Liang and Pardee [ Science
, 257, 967-971 (1992)]. Therefore, the sequences of both the 10mer and the
new 11mer oligonucleotides were determined by a statistical evaluation of
species-specific coding regions extracted from the EMBL database. The 11mer
primers used for reverse transcription were selected for localization in
the 3'-region of the bacterial RNA. The 10mer primers preferentially bind
to the 5'- end of the RNA. None of the primers show homology to rRNA or
other abundant small RNA species. Randomly sampled cDNA bands were checked
for their bacterial origin either by re-amplification, cloning and
sequencing or by re-amplification and direct sequencing with 10mer and
11mer primers after asymmetric PCR.
ARTICLES
Primer design for a prokaryotic differential display RT-PCR
Institut fur Medizinische Mikrobiologie, Universitat Rostock, Schillingallee 70, D-18057 Rostock, Germany. rainer.fislage@medizin.uni- rostock.de
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