dtcroke at iol.ie (David T. Croke) wrote:
>>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.
You could try the method of Welsh. It uses just one primer for the RT and PCR steps. This means that you are amplifying fragments at any distance from the 3' untranslated. If you are interested I've optimized this method, and the protocol worked in my hands and in at least 5 other labs. If you want to give it a try, I can e-mail the protocol.
Hope this helps.
Department of Biological Chemistry
The Hebrew University of Jerusalem.