Hello everyone! I recently have a difficult question regarding T-DNA insertion lines and I am looking for help!
I ordered a SALK line which according to the website, the T-DNA was inserted into its 2nd exon. After genotyping, I selected the homozygous lines.
To proceed the experiment, I am trying to do semi-quantity PCR to check the transcription level of the gene. However, even for the homozygous lines, the gene (CDS, to be more specific) can sometimes still be detected. Has anyone also confront such situation before? How to explain the result?
PS: DNAse was added. 40 cycles for semi-quantity PCR.
Hi Zeya
probably the protein is not produced anymore due to this insertion, but the mRNA is transcribed and therefore detectable by qPCR.
all the best
fred
Ah yes, this question reminds me of complications in my thesis.
Top possibility, the information from Salk is not accurate. Many/most of the T-DNA insertions are NOT present in the estimated location shown on the website. In fact, a good percentage aren't even present at all and are an artifact due to cross-contamination. The only way to know the location is to sequence the junction between the T-DNA and the insert site.
Don't bother with qPCR until you have sequenced the junction on both sides. And double-checked that you don't have a chromosomal translocation (see below).
Another very common possibility, your primers are not unique to your transcript of interest. Most Arabidopsis genes are in gene families and have a LOT of sequence similarities.
Another possibility is that you get gene expression driven by a promotor in the T-DNA insertion. Rather common in fact.
Finally, about 20% of T-DNA lines from the Salk collection have chromosomal translocations associated with the T-DNA insertion site. The only way to know FOR SURE whether you have one is DNA mapping. See the attached paper.
Good luck!
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