DYE PRIMER vs. DYE-TERMINATOR

Peter Myler mylerpj at u.washington.edu
Wed Feb 15 14:41:08 EST 1995


On Wed, 15 Feb 1995, Mick N. Mulders wrote:

> The following problem I have encountered and would like to hear your
> comments about:
> 
> I have amplified RNA (polio) in two different fragments with three primers.
>  both fragments have therefore the same 3'-end. (630 vs. 290 bp). when I
> compare part of the overlap, which I could sequence in both directions, I
> stumble across sequence differences between both fragments, which should be
> the same. the smaller fragment was sequenced with the dye-terminator
> protocol, the larger fragment with dye-primers. did anybody actually
> encounter differences in basecalling using the the different protocols? or
> could anybody give me references to publications discussing this or related
> topics?
> 
> help would be appreciated VERY MUCH as I am in the process of finalizing a
> manuscript.
> 
> please send comments also to my e-mail account, as the turn-over within the
> newsgroup is higher than my frequency reading it.
> 
> 
> Mick (mulders at rivm.nl)
> 
> 

The differences may very well be due to compressions in the dye primer 
reactions - it does not routinely use dITP, which is used in 
the dye terminator reaction, and hence is more likely to give 
compression.  Close inspection of the gel tracing should show evidence 
for compression.  

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Peter J. Myler                                 phone: (206) 284-8846x332
Seattle Biomedical Research Institute          FAX: (206) 284-0313
4 Nickerson Street                             e-mail: MYLERPJ at U.WASHINGTON.EDU
Seattle, WA  98109-1651
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