[Arabidopsis] T-DNA insertion site

Bernd Weisshaar via arab-gen%40net.bio.net (by weisshaa from cebitec.uni-bielefeld.de)
Tue May 22 06:41:44 EST 2012


Dear Michelle,

the prediction of T-DNA insertion positions is not really exact if one has in hand only the FST (flanking sequence tag) which is generated by PCR from the insertion line. For this reason, the data you find in SIGnAL / T-DNA Express, TAIR or in the SimpleSearch database are only PREDICTIONS.

At GABI-Kat, we do in-house confirmation of the predicted insertions. 
http://www.gabi-kat.de/
The confirmation process includes PCR amplification of an amplicon that spans the insertion position (fusion point of T-DNA and genomic DNA at one of the two "borders" of the inserted DNA), and sequence analysis of this amplicon. For the lines that have been confirmed (and which you can order from NASC), these confirmation sequences are available from SimpleSearch. Not always, but in quite some cases, the detailed inspection of these confirmation sequences does allow to deduce the insertion position quite exactly. However, one should keep in mind that data about the other border are usually not available, and it might be that some (several) nucleotides are deleted between the two fusions points.
Example:
http://www.gabi-kat.de/db/getseq.php?filename=19-K018911-022-250-D07C-DL91

The initial prediction of the insertion position can turn out to be quite inaccurate, and in (very few) extreme cases (mostly when paralogous loci are involved) the insertion can turn out to be located in another gene on another chromosome.

Best regards, Bernd Weisshaar


On 19.05.2012, at 01:24, Michelle Tjahjadi wrote:

> Hi all,
> 
> Is there a way to find the specific site of a T-DNA insertion? I'm looking
> at several SALK, SAIL, and GABI lines. TAIR, T-DNA Express, and the
> respective company websites only seem to provide the flanking sequences.
> Using the SALK T-DNA primer website, SALK can provide LP and RP primers to
> confirm the insertion. When I search for these primers, I get the sequence
> of each primer, and there is a segment that states, ex: "Insertion chr1
> 8999832". At first, I thought the number corresponds to the exact gene
> number at the insertion site, but now I'm thinking it's the flanking
> sequence. SALK also states that the insertion can be up to 300 bp away from
> the flanking sequence, so I really think the gene number is site of
> flanking sequence. Does anybody know for sure? Besides ordering the stock
> and doing the actual PCR myself, is there another way to find the exact
> location of a T-DNA insertion online?
> 
> Thanks,
> Michelle
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Bielefeld University, Department of Biology
Prof. Dr. Bernd Weisshaar
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