What is TOGA ?
dspinella at chugaibio.com
Wed Jul 1 10:37:26 EST 1998
> Does anybody know the method to detect gene expression called "TOGA" (Total
> Analysis of Gene Expression by Automation) used by Digital Gene technologies,
> La Jolla. I have read about it but it was not enough to get full picture.
> Thanks a lot.
> Narendra Kaushik
In the last version that I have heard about, TOGA works by generating a
3' end directional cDNA library. I believe they use a biotinylated
Not1-dT primer to produce cDNA followed by MspI digestion and capture of
the 3'-most fragments on streptavidin beads. This material is then
released from the beads with NotI digestion and cloned into the Not/ClaI
site of a standard vector like pBluescript. cRNA is then run off using
a T3 or T7 promoter sequence upstream of the cloning site. Supposedly,
the relative abundance of cDNAs in the library is maintained in the cRNA
pool. The cRNA is then subjected to PCR amplification with an antisense
primer corresponding to a 3' vector sequence and a panel of 256
different sense primers. Each sense primer corresponds to 5' vector
sequence, the MspI cloning site (CCGG) and all 256 possible 3'
tetrameric sequences. Each of these primers (when paired with the
common antisense primer) would amplify roughly 1/256 of the cRNAs in the
pool, generating hundreds of PCR products of varying sizes. Performing
PCR with all possible primers and running the products in different
lanes, gives a gene expression profile -- wherein the quantity of PCR
product is proportional to the steady-state message level of the
transcript from which it was ultimately derived. That's the theory
anyway. I'm sure you can contact DGT for more info (http://www.dgt.com)
Hope that helps.
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