PCR problems with fluorescent labeled primers

jaeger jaeger at pilot.msu.edu
Wed Feb 24 20:46:32 EST 1999


Dear colleagues,

I am working on the genetic analysis of red pines. Simple sequence
repeats
(SSRs), also known as microsatellites or short tandem repeats have
proven
valuable as genetic markers. Allele sizes differ by length of the repeat

unit and markers can be amplified by PCR from small amounts of DNA.

And now my question:
I use flourescent labeled forward primers and unlabeled reverse primers.
By
testing these primers in PCR by amplifying several DNA samples I didn't
get
in most cases a PCR product. On the contrary by using the same forward
primers (same sequence), but unlabeled I got in most of the cases a PCR
product.
I used a touchdown amplification protocol, the first two cycles included
a
denature step at 94  C for 1 min, an annealing step at 60 C for 1 min
and an
extension step at 70 C for 35 s. 18 cycles consisted of a denaturing
step at
93 C for 45 s, an annealing step at 59 C for 45 s (which subsequently
was
decreased by 0.5 C every cycle until a final temperature of 50 .5 C was
reached), and an extension step at 70 C for 45 s. Conditions for the
last 20
cycles were 92 C for 30 s, 50 C for 30 s, and 70 C for 60 S, followed by
a
final extension at 70 C for 5 min.
Since I didn't  have such a lot of problems to get a PCR product with
the
same , but unlabeled  forward primers, I assume the label causes the
problem. The primers are labeled with HEX, 6FAM, TET and NED.

I appreciate if someone could give me an advice to solve this problem.

Rosemarie


Rosemarie Walter
E-mail:  walterro at pilot.msu.edu




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