BIONET DNA Sequencing Survey
hamel at ccu.umanitoba.ca
Mon Jun 21 12:45:18 EST 1993
In article <C8vqIC.KF8 at mentor.cc.purdue.edu> pmiguel at bilbo.bio.purdue.edu writes:
>In article <19JUN199308260889 at aardvark.ucs.uoknor.edu>, broe at aardvark.ucs.uoknor.edu (Bruce Roe) writes:
>>I have a tendency to think about bands in all 4 lanes as being due
>>to "template compression" i.e. secondary structure, since "product
>>compression" also is due to secondary structure ( fold back )
>>in the synthesized strand.
> Yeah, but calling template secondary structure "compression" is
>confusing. "Product compression" is seen as bands compressed together on
>the sequencing gel, thus the name. Extremely bad compression could look
>like or be accompanied by what you call "template compression" -- bands
>across all four lanes. I call this "fall-off" because it is so refered to in
>the Sequenase manual, because it makes sense to me and, probably more
>important isn't easy to confuse with "compression."
> I'm not just quibbling over nomenclature here. If you have a compression
>you need to run a gel with formamide in it or do rxns with dITP or dazaG
>replacing dG. If you have fall-off you need to increase the temperature of
>your extensions or use SSB to eliminate template secondary structure.
Also, "premature terminations ... or falloff" can be removed from areas
where they occur on seq gel simply by adding TdT labelling mix as step
after ddNTP steps ... causes "fall off" products to be elongated (since
they likely contain hydroxyl group at 3' end), hence migrate higher up in
gel ... if TdT is need for "fall off" regions that appear further from
primer (either sequence from other end if possible) or include
pyrophosphatase in the ddNTP rx'ns, along with the TdT, this ought to help.
Compressions (several bands in same lane being "squished" together, often
resolve in presence of 7-deaza dGTP in labelling AND ddNTP rx'n mixes
(tends to work better than use of dITP ... at least with Sequenase) ...
compressions may still occur with cycle sequencing ... likewise 7 deaz
dGTP is easiest route to start with, then if still no improvement perhaps
formamide in seq gel may be warranted ... also, subclones containing different
region frequently overcomes compression problems, for example, subclone
beginning right at compression zone versus subclone where compression
causing zone is more in middle ...
It would have been nice to have FAQ on this particular topic years ago ...
with so many capable sequencers out there using this newgroup, perhaps a
constantly "evolving" FAQ/archive could be developed? (or is there one in
existence already, and I was just too lazy to search the Internet for it?)
Andre Hamel email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services lab tel.: (204) 945-7630
Infectious & Genetic Disease FAX: (204) 945-8062
Mol.Biol.Lab., Winnipeg, Manitoba, CANADA
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