Fluorescent Nucleotide Analogs

[KATE at BIO.TAMU.EDU]

On October 5, 1993, <ottcr at esvx17.es.dupont.com> wrote:

> Is anyone interested in trying some new fluorescent 
> nucleotide analogs for fluorescence in situ hybridization, 
> point mutation analysis or genome mapping?  
                                     
This posting brought a swift rebuke for commercial advertising on 
the network, which snuffed out further discussion.  But the 
information raised interesting questions in my mind.  We are just 
starting to use in situ hybridization, and I have wondered about the 
relative merits of using radiolabeled probes, vs. digoxygenin labeled 
probes, vs. probes incorporating fluorescent nucleotide analogs.  Are 
the latter adequate for detecting mRNAs in tissue?  If a digoxygenin-
labeled riboprobe is used, followed by fluorescein-tagged anti-
digoxygenin, is sensitivity improved?  Is resolution diminished?  
When using digoxygenin-labeled probes, is the signal amplified by 
incorporating an additional antibody step into the protocol (e.g. 
sheep anti-digoxygenin, followed by rabbit anti-sheep/FITC)?  

Responses from more seasoned investigators would be welcome on any of 
these points.  If they are directed to me, I will sort and edit the 
responses, and repost.  Thanks!

Kate VandenBosch
Department of Biology
Texas A & M University
College Station, TX 77843
FAX: 409-845-7707
INTERNET: kate at bio.tamu.edu