opinions on auto sequencers

Alexander Kraev kraev at bc.biol.ethz.ch
Thu Jun 27 10:50:21 EST 1996


Richard Hardy wrote:

>In our experience (about 1000 sequence runs/month, ABI 377 and 373), the
color blobs result from incomplete cleanup after the cycling reaction.
Cleanup can be either by spin-column or (using the more recent ABI cycle
sequencing kit) precipitation.  The only time we see blobs is occasionally when the template doesn't generate sequence, so maybe it
results from large amounts of leftover fluorescent components that the standard cleanup won't remove(?)...<

Thank you for mentioning that, because the removal of unincorporated
label seems to be a major problem DURING TRANSITION from radioactive
to fluorescent sequencing. However, my original response was to the
statement that four-colour chemistry is inherently simpler than the
one-colour. With one-colour chemistry using labelled primer one can
load directly the cycling reaction, while the same reaction with
fluorescent dNTP may require a cleanup. So, it is not simplicity of the chemistry that distinguishes ABIs from other instruments, but rather a throughput AND the price difference. Finally, ABI's "user-friendly" ready-reaction mixes are horribly expensive (at least here in Switzerland), but the local representatives make every effort NOT
to let you make your  own kit at a lower price (where can one  buy ABI's fluo-ddNTPs?). In contrast, with IRD41- and Cy5-dNTP one can use different polymerases, including those that are not yet on the market...

Alexander Kraev, PhD                     Internet:    kraev at bc.biol.ethz.ch
> Lab.of Biochemistry III                            Phone:   0041-1-632-31-47
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