Help with tDNA insertions and RT-PCR

Dietrich, Charles CDietrich at danforthcenter.org
Wed Dec 1 16:06:15 EST 2004


I am attempting to do some Quantitative Real Time PCR on various 
homozygous Arabidopsis t-DNA exon insertion alleles for one gene.  I 
am finding that my confirmed homozygous lines give Ct values the same 
as wild-type with gene specific amplicons except those that directly 
flank the insertion site which, as expected, fail to amplify.  I 
would expect other amplicons in my mutated gene - though not directly 
disrupted by the t-DNA, to be drastically reduced in abundance 
because the gene would be producing at best aberrant transcripts that 
would be degraded rapidly.  I would expect to be able to see this 
difference on the Real Time machine.

Is this a common phenomenon with Arabidopsis tDNA insertion alleles 
or is this unique to this gene?  This is a problem mainly because I 
was attempting to use the same Taqman amplicon for multiple alleles 
with insertion sites in different parts of the gene.  I now am using 
SYBR green because I don't want to design Taqman probes specific for 
each allele.

I am just wondering if this is common in Arabidopsis.  From my 
previous work with Maize transposable elements I know that a 
transposon insertion typically reduce the transcript to near zero for 
the entire gene because they cause a block in transcription.  I 
assumed a tDNA in an Arabidopsis exon would also block transcription. 
 Was this a bad assumption?

Thanks for you input.

   


Charles R. Dietrich, Ph.D.
USDA-ARS
Donald Danforth Plant Science Center
975 North Warson Road
St. Louis, MO 63132
Email: cdietrich at danforthcenter.org
Phone: 314-587-1466
Fax: 314-587-1566 





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