We've been using DDRT-PCR to study differences between colon tumour
cell-lines of various types +/- antisense oligonucleotides directed
against a number of cytokines which the cells are known to produce. After
some optimisation (which in our hands largely seems to depend on the
10-mer concentrations used in the amplification) we have now subcloned &
sequenced a number of difference products in the 150-350 bp size-range.
The clones show weak homology to a number of items in genbank but I am
afraid that we are basically just pulling out bits of 3' untranslated
region from our target cDNAs. If anyone has any suggestions as to good
methods for pulling out longer cDNAs corresponding to our difference
products I would be very grateful!
David T. Croke <dtcroke at iol.ie; dtcroke at rcsi.ie>
Molecular Biology Laboratory, Royal College of Surgeons in Ireland,
St. Stephen's Green, Dublin 2, Ireland.
Tel.: +353-1-402-2131; Fax: +353-1-402-2467.