Talk:Digoxigenin
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It can easily be attached to oligonucleotides, at either 5' or 3' ends by chemical modifications.
To detect mRNA production in a given cell :
1- Prepare complimentary RNA with DIG attached to them. Nowadays, you order that over the internet.
2- Following your preferred protocol for In situ hybridization, attach this probe to mRNA produced by the cell.
3- Use an anti-DIG antibody to detect DIG (and hence, mRNA).
This is basically rubbish. I'm not sure how Dig is attached, but it's usually conjugated to a specific nucleotide (normally Uridine) which is incorporated into the complimentary mRNA when it's synthesised - therefore attaching it to the bits where nucleotides join together into strands is unlikely.
Nobody in their right mind would order RNA over the internet; everyone but everyone produces their mRNA fragments in house. Presumably whoever wrote this is getting confused with primer design, which is (as far as I'm aware) largely done online; given oligonucleotide primers (10-20 nucleotides) and a cDNA library or a complete set of genomic DNA, you can produce a large number of gene fragments (commonly 100-1000 nucleotides) using PCR; which is usually put in a plasmid. You then use an RNA polymerase, and a nucleotide mix containing only Dig-labelled Uridine triphosphate, to make a transcript that can be detected by antibody.
It can also be conjugated to specific sugars and allowed to incorporate into glycoproteins in live cells, and detected by anti-Dig antibodies.
Am updating to reflect this, and to make it read less like a protocol for ISH.
Confuseddave 20:03, 12 January 2007 (UTC)
