?RNA 3'-end labeling/chain termination
rpierce at u.washington.edu
Thu Apr 20 15:29:47 EST 1995
We're trying to block RNA-self priming by adding a chain terminator prior
to cDNA synthesis. Two enzyme systems (and two chain terminators) come to
mind: 1) Yeast Poly A polymerase and Reverse transcriptase with
Dideoxy nucleotides and cordycepin-5'triphosphate.
Does anyone have any experience with these systems? Specifically, I'm
interested in the following:
1) Can the two enzymes be combined in a single reaction?
I have been
told that RT is more 'tempermental' than Poly A Pol - - that the Mg
concentration is critical for optimal RT function. The Poly A Pol buffer
specifies Mn -- - will
Pol A Pol also work wwith Mg and if so, will the properties of Poly A Pol
change with Mg instead of Mn?
2) Can the two enzymes utilize the same terminator nucleotide with
I have been told that Poly A Pol can
not utilize dideoxy because of it's 3 and 5 deoxy condition. Cordycepin
on the other hand is onlly 3 deoxy and can be used by Pol A. Is this
true and could RT use Cordycepin with 'workable' efficiency?
I hope someone out there knows the answer.
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