?RNA 3'-end labeling/chain termination

Robert Pierce 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 
'workable efficiency'? 


 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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