dye-terminators and sequencing

Jackson Shea sheaj at ohsu.edu
Wed Oct 19 17:02:26 EST 1994


Hi users...
In response to the questiuons/issues raised by "igloi at oligo..."
1] Single-fluor (FAM) ddNTP's are available from duPont/NEN. I've used
them on the ALF - but not for traditional sequencing, for alternative
method development.... and yes, they showed up, very nicely.

2] Talk to Pharmacia about how-to-do modifications on the ALF to run water
through a chiller and thus run the machine at 4-10 degrees C. This is how
we do SSCP on the ALF. 

3] Internal labeling w/ FdATP works well _if_ you have an even - or high -
level of "A's" in the region to be sequenced. And if you have enough
template to use T7 DNA polymerase. If you have a GC rich/ repeated region
to analyze, there are probably less stressful approaches than to try to
use FdATP.

_Now_ - for my question of the week.... has anyone used, successfully,
ABI's dye-terminator kit for ss DNA to do solid-phase [bead] sequencing of
genomic DNA's - to detect heterozygousities?
 I get _great_ results on the ALF, but I have a three-week backlog, so
"the powers-that-be" would like to try things on the ABI...higher thru-put
and all....[Sorry, Casey and Trace] There is supposed to be a paper that
addresses this very issue in "Methods in Enzymology" vol 218(?), by
Schofield, Jones, and Vaudin. I've looked in 7 libraries at the TMC and
several departmental conference rooms for this volume and paper, but to no
avail. If anyone has access to this paper and/or personal experience with
*dd genomic sequencing on the ABI please contact me via FAX or email:
Inet:maryic at imgen.bcm.tmc.edu   (that's in Texas, CDT, y'all.)
FAX:713.798.7383
voice:713.798.6548

Thank you very much,

Mary Coolbaugh Murphy

{sorry about sending this through you, Jackson, we've had a lot of
flooding around the Med Center and some of our net functions are still a
little "chaotic"...things like trying to post to net-news, etc.....}




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