IUBio

Dye-term Sequencing of crude lysates

Shaun Tyler styler at hwc.ca
Sun Nov 3 18:30:28 EST 1996


> From: Jack Leonard <Jack.Leonard at amicon.com>
>=20
> Subject: Dye-term Sequencing of crude lysates
>=20
> A recent article in Biotechniques 21:453-457 by Chen et al. (Sept
> 1996,
> "Automated DNA Sequencing Requiring No DNA Template Purification")
> reports the use of "hot start" in thermal cycling for automated DNA
> sequencing reactions to obviate plasmid DNA template purification, and
> to generate up to 500 bases with insignificant background from CRUDE
> LYSATES. Is anyone familiar with this technique, and if so, do they
> agree with the authors that the overall quality of DNA sequence is
> considerably improved and that it is becoming unnecessary to improve
> the
> quality of DNA templates (i.e., use DNA directly from E. coli without
> purification)? Thank you greatly for your
> response in advance.
>=20
> --
> Jack T. Leonard, Ph.D.
> Manager, Lab Products and Applications
> Amicon, R&D


Hi-Jack (sorry but I couldn't resist),

I haven=92t seen the article you referred to but at the last ABI Users=20
meeting I attended (in Ottawa) a presentation was made by a guy named=20
Qifan Chen (????) describing this approach.  The data he presented was=20
pretty impressive.  The people running the sequencing facility at the=20
same institute seemed to agree and had used this technique a number of=20
times with equal success.  Personally, I have only tried the technique=20
once and was satisfied with the results.  I can=92t really comment about=20
any claims that the data is better than using purified plasmid but it was=
=20
at least as good.  The only reason we haven=92t adopted this method (beside=
=20
the fact that we don=92t normally do the template preparation) is that I=20
found it somewhat time consuming in both hands-on and overall preparation=
=20
time.  The increase in hands-on time was in part due to the use of the=20
hot start and the added manipulations it required.  If this step could be=
=20
omitted and the reactions set up as usual (we generally use the Prism=20
dye-terminator kits) the technique might not be so bad.  The increase in=20
overall preparation time was mainly due to the prot. K incubation;=20
however, if you were doing a large number of templates this might balance=
=20
out.  We don=92t normally prepare large numbers of templates at any one=20
time (usually just enough to fill a gel) and I found that in the time it=20
took me to prepare the crude lysates I could have easily prepared 2 or 3=20
times the number of samples using a kit.  There are two big advantages=20
that I can see with this technique.  The first is that it is CHEAP.  If=20
you are not overly concerned with labor costs (e.g. you have access to a=20
grad. student) then this route may be attractive.  The second is that if=20
you have the necessary equipment the technique is readily adaptable to=20
automation.  We are in the process of acquiring a robotics workstation=20
and if we get it I plan on taking a really serious look at routinely=20
implementing this approach to sample preparation.  We still find that=20
when using plasmid purification kits there is definitely a difference in=20
the quality of the sequence data depending on the manufacturer of the kit=
=20
and frankly I'm still surprised that this technique works as well as it=20
does.

I hope this answers some of your questions :-)


Shaun Tyler
DNA Core Facility
Laboratory Centre for Disease Control
Health Canada

Ph#:  (613) 941-6441
FAX#: (613) 957-1358

E-mail:  styler at hpb.hwc.ca




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