Primer Testing: Its Worth the Wait

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Primer Testing Its Worth the Wait
We have found the Primer Pre-Screen to be an
invaluable step for Maize TILLING. We simply
verify that the primers amplify a single product
of the expected size and sequence from our B73
and W22 TILLING inbreds. The primer pre-screen
identified 55 primers that would not have TILLed
well for a variety of reasons as shown on the
graph below. We have used 69 primers sets in
TILLING, and only 5 were deemed failures 3 were
TILLed before the pre-screen was implemented, 1
has a GC-content gt 70, and 1 did not amplify
enough product under our TILLING PCR conditions.

Primers that pass the Pre-screen are TILLed
successfully gt 90 of the time!
One reason primers fail is that the PCR product
amplified is gt 1.5 kb (our upper limit), usually
due to introns that the user did not know about.
Targets with a run of a single nucleotide (ex 10
As) result in two sequence traces, one offset by
a base, that are difficult to interpret in the
context of identifying mutations. If your primers
fail, we will send you an email that includes 1)
the reason(s) they failed, 2) any suggestions we
may have for your next primers and 3) the
sequence data we have generated from B73 and W22.
To date we have received 92 TILLING requests, 55
from the Public and 37 from our beta-test. The
time to process a request has varied from 3 - 10
months. We have found that users who test primers
on their own have their screening request move
through the TILLING pipeline more
quickly. Primers can get held up in the
Pre-Screen due to 1) repeating the pre-screen to
be certain about the PCR and/or sequencing
results or 2) the primers were failed but the
user has not re-ordered another set of primers.
GC-rich targets have taken longer at the TILLING
stage as we battled with poor quality TILLING
gels. Screening requests can hit another
bottleneck at mutation sequencing, especially
when we TILL many targets within a short time.
With our current population, we have completed 10
screening requests. Once we add new mutants to
the population we will resume screening of the 35
orders that have an incomplete allelic series.