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
