ABI 373A DNA Sequencer data just fades away.

Paul Morrison p_morrison at dfci.harvard.edu
Mon Feb 6 14:45:35 EST 1995


Here is a problem that is starting to really bug me. It has been
occurring intermittently since Oct94 at an increasing rate. Just as
soon as we think we have figured out the problem. Whap, it's back. So a
brief description of the problem.

"Core Facility" (different sample preps) templates sequenced double
stranded, Taq polymerase, cycle sequencing, DYE labeled terminators.
On occasion we will get a gel that has _something_ wrong with it. We
now call them "fast-fade" gels. On these fast-fade gels many lanes of
data will look good but then abruptly (within 20 nucs) fade to
practically nothing. These problem lanes will usually (but not all)
come in groups on the left or right hand side of the gel. This problem
has occurred in various places. After 50 to after 400 nucs of good
data. We have contemplated just about every reason and not one of them
holds up. Because it happens infrequently and we are sequencing Core
Facility DNA from various sources this has been very hard to track
down.

What the problem looks like on the:

Analysis file. Peaks are great and then abruptly drop and get the
"shakes". There is usually trailing _in front_ of the peak that happens
as the peaks spread out and quickly change to shaky waves of color.
Raw Data file. Peaks are great and then fade to nothing.
Gel file. Peaks are great and then get blurry. It looks as if the same
amount of color is there but the resolution has gone to pot.

Reasons why we have ruled out:

secondary structure:
the problem has occurred sequencing the ABI standard which shows no
secondary structure problem. Also templates will be rerun the next day,
same conditions and the problem disappears.

Template prep, primer prep:
templates from different sources fail and as above work fine the next
day.

The Gel:
We have replaced all reagents many times. No correlation. 2 gels made
at the same time by the same person loaded on different 373's. One
fast-fades, one is fine. (This happened last night after we thought we
got it).

The sharkstooth comb:
We prep this the same way. No loose acrylamide, no problems.

Sample buffer, the formamide, the blue juice:
Replaced, brand new, deionized. Still happens.

The centrisep columns:
Same lot, same technique used, one good gel, one bad gel.

The Taq enzyme, the terminator dyes:
The same lot, the same tube, one good gel, one bad gel.

The 373:
We have 2 373's. Old373 got a stretch upgrade in Apr94. New373 was
delivered as stretch Sep94. The problem has occurred on both. While it
is true that the problem has only occurred on a stretch machine, we ran
stretch gels from April to October with no problems. The problem has
occurred only once on the new 373 and has occurred 8-10 times on the
old but I feel this could easily be _not_ significant because the old
one is used more frequently.

Data collection:
The drop off does not occur at exactly the same spot (scan line) in the
gel file. It will happen in _around_ the same spot but will have enough
variation that rules out data collection problems.

Final thoughts:
Because it doesn't happen often it has been very difficult trying to
pin this down. It may have multiple reasons and they all have to be
_below_ tolerances in order for it to show up. If this is the case then
it may be a reagent or column spec. that has changed since October 94.

Has any other lab seen this problem? Anything close to this problem?

Thanks for any help or suggestions.  -Paul

Paul Morrison   Dana1030
Molecular Biology Core Facilities
Dana Farber Cancer Institute
44 Binney Street
Boston, MA 02115

p_morrison at dfci.harvard.edu



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