PCR and Agarase
ldow at biosys.apldbio.COM
Fri Apr 2 10:54:16 EST 1993
In article <C4tBD8.CJ7 at ccu.umanitoba.ca> hamel at ccu.umanitoba.ca (Andre Hamel) writes:
> This topic on agarase/agarose DNA band:inability to PCR arouses my
>in a nutshell, perhaps serially diluting in water and/or formamide
>and/or adjusting the salt/Mg++ conditions might be useful?
Disclaimer: I work for Applied Biosystems.....not New England Biolabs, nor
do I have any interest in agarase other than as a consumer.
I was actually wondering this myself since I have had good results when
using the NEB agarase as a method to purify my PCR products prior to
sequencing them. I have been using this as a control method to look
at the effects of smaller sized products on PCR sequencing. I have also
been sequencing in parallel products purified by spin column and by
magnetic purification. I have not seen any differences that I could
attribute to the agarase.
However, I have not tried any subsequent PCR amplification of the purified
product. The buffer and salt concentrations that we use for sequencing (I am
speaking of Taq cycle sequencing on an ABI fluorescent sequencer) differ
from those used in the Perkin-Elmer Taq buffers:
The PE PCR II buffer is (at 1X concentration) 50mM KCL, 10mM Tris, pH8.3
at RT, and usually 1-3mM MgCl2
The ABI Taq Cycle Sequencing Buffer is (at 1X concentration)
40mM Tris pH8.9 at RT, 10mM Ammonium Sulfate, 2.5mM MgCl2.
We also use quite a bit more enzyme for sequencing that is used in
PCR, we use close to 8 enzyme units for a set of 4 termination reactions,
at a concentration of approx. 0.2 EU/ul. This might be the critical difference.
If the agarase is inhibiting the enzyme it would be much less likely
to be observed in our sequencing system than in a PCR reaction where the
concentration of the enzyme is 10 times less. I am assuming here that generally
one uses 2 EU of Taq per 100ul PCR amplification, which gives a concentration
of .02 EU/ul.
>Have you tried serially diluting in H2O or TE then boiling the sample
>for few minutes AFTER agarase digestion and/or modifying the Mg++ and/or
>salts (KCl) conc. of the pcr rx'n?
>Wondering whether or not the digested agarose/DNA/agarase mixture might
>contain some chelating and/or salt conc. effects that can affect the pcr.
>Looking at the Beta agarase I descriptions (page 93 of New England Biolabs
>1992 catalogue) I see nothing in particular about the enzyme storage
>buffer, nor the reaction conditions (soak gel in 10x vol. of TE, wash,
>dilute, melt, etc) that would appear to be inhibitory toward pcr. Perhaps
>the grade of agarose used? Highest grade LMP agaroses (for instance from
>Biorad or FMC), might they not be okay? I'd suggest avoiding use of NuSieve
>or any other agarose that are NOT too pure (too much sulfur, other metals,
>such as Zinc, Calcium?)
Leslie Johnston-Dow ldow at apldbio.com
Applied Biosystems Inc
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