Nucleic Acids Research, Vol 27, Issue 5 1345-1349, Copyright © 1999 by Oxford University Press
Y Mizuno, P Carninci, Y Okazaki, M Tateno, J Kawai, H Amanuma, M Muramatsu and Y Hayashizaki
We have developed a method for high-efficiency window separation of cDNA
display by increasing the specificity of priming in reverse transcription.
In the conventional method, two-base anchored oligo(dT) primers
(5'dT16VN3', where N is any base and V is G, A or C) are used to make
windows for the display of transcripts. However, reverse transcriptase
often extends misprimed oligonucleotides. To avoid mispriming from dT16VN
primers, we have developed two new technologies. One is higher temperature
priming with reverse transcriptase thermoactivated by the disaccharide
trehalose. The other is the use of competitive oligonucleotide blockers
that hybridize to the non- selectively primed mRNAs, preventing the
mispriming from the VN site. These methods were combined to improve
restriction landmark cDNA scanning (RLCS), resulting in the elimination of
the redundant signals that appear in different windows. This was achieved
by the increased specificity of initiation of reverse trans-cription from
the beginning of poly(A) sites. This method paves the way for the precise
visualization of transcripts to allow expression profiles in individual
tissues and at each developmental stage to be understood.
ARTICLES
Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display
Laboratory for Genome Exploration Research Project, Genomic Sciences Center (GSC), Institute of Physical and Chemical Research (RIKEN), Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074, Japan.
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