PCR and Agarase

Andre Hamel hamel at ccu.umanitoba.ca
Thu Apr 1 11:05:32 EST 1993


	This topic on agarase/agarose DNA band:inability to PCR arouses my
curiosity:
in a nutshell, perhaps serially diluting in water and/or formamide
and/or adjusting the salt/Mg++ conditions might be useful? 


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?)

Another thought. Since some thermostable DNA pol. can withstand formamide
up to 20% (final conc. v/v), for instance, Pfu, Tth, Vent polymerases,
what would happen if a small portion of the agarase digested gel, say 10
uL, was added to, say 100 uL formamide, boiled, then serially diluted in
water or TE, THEN small portions (say 1 to 10 uL) added to 50 uL pcr
rx'n mixes then amplified? Might the formamide somehow chemically
"sequester/solubilize or otherwise modify/interact with" any of the "free"
agarose digestion products (neoagaro- oligosacch.)? I know they ARE soluble
in water, likely so in formamide, but perhaps "something else" might
"happen"? Of course, if formamide is used, the temperatures of each cycle
step would have to be appropriately decreased, say by 0.5oC for every 1%
formamide. 

:-)

Andre Hamel
Manitoba Veterinary Virology
Winnipeg, Manitoba
Canada

email: hamel at ccu.umanitoba.ca


In article <1993Mar31.174052.8440 at mbcf.stjude.org> neale at mbcf.stjude.org writes:
>In article <1993Mar8.160923.27433 at gserv1.dl.ac.uk>, r.nakisa at ic.ac.uk writes:
>> Dear Netters,
>> 
>> This is to confirm that DNA gel-purified with beta-agarase can't be
>> amplified by PCR.  I did a control experiment with a template and
>> primers that ALWAYS work in which I loaded the template onto a LMP gel
>> and purified it with a beta-agarase digestion.  There was no
>> amplification.
>> 
>> I have just spent a month trying to amplify my ligation product. 
>> Because of a sin of omission in the agarase instructions the
>> amplification was doomed not to work :-(.  The instructions say
>> 
>>    Beta-agarase I acts by cleaving carbohydrate bonds, freeing trapped
>>    DNA, and producing carbohydrate molecules which can no longer gel. 
>>    The remaining carbohydrate molecules and Beta-Agarase I will not, in
>>    general, interfere with subsequent DNA manipulation including:
>>    restriction endonuclease digestion, ligation and transformation.
>> 
>> And, of course, excluding PCR.
>> 
>> Someone has written to me telling me his lab has found exactly the same
>> thing with the american version of the enzyme called gelase.  I'm rather
>> miffed that I wasted so much time for nothing.
>> 
>> Others, beware!
>
>
>
>G'day all,
>
>Just thought I'd add some further anecdotal evidence regarding agarase and
>enzyme behaviour. We had been purifying DNA fragments for probes using agarase
>and found that over time the DNA no longer labelled sufficiently to be used in
>hybridizations, or worse gave very high backgrounds.
>
>We had not had that problem prior to using agarase purification of probes. We
>have now returned to electroelution or gel spin technique.
>
>Regards,
>
>
>Geoff
>
>
>*************************************************************************
>Geoffrey Neale Ph.D
>neale at mbcf.stjude.org
>
>Dept. of Virology and Molecular Biology
>St. Jude Children's Research Hospital
>Memphis, TN
>Voice: (901) 522-0400
>Fax: (901) 523-2622
>*************************************************************************
>
>"Such is life" - Ned Kelly, infamous Australian bushranger before his hanging
>
>
>
>
>



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