neale at mbcf.stjude.org
neale at mbcf.stjude.org
Mon Apr 11 17:18:53 EST 1994
In article <-110494115337 at dent-226-97.dentistry.umich.edu>, @umich.edu (Steven Coon) writes:
> In article <sticknbd-080494070634 at 220.127.116.11>,
> sticknbd at ctrvax.vanderbilt.edu (Henry) wrote:
>> When I make random-primed probes, the protocol I use
>> says to stop the reaction after 30 to 60 minutes with
>> EDTA (or heat), which I always do. What would be the
>> consequences of allowing the reaction to continue? I
>> cannot envision any ill effects of letting the reaction
>> go, even if it were to go on for several hours. Comments?
>> sticknbd at miranda.cc.vanderbilt.edu
> I think that the enzyme doesn't last for very long anyway so it really
> doesn't matter very much. In most cases the majority of labeling occurs in
> the first 15 minutes anyway.
> Steve Coon
> sdcoon at umich.edu
> University of Michigan Medical Center.
In answer to the first query: it should not matter "in theory" if you let the
reaction proceed longer. In practice, the Klenow supplied with your protocol
may be contaminated with exonuclease, and therefore prolonged incubation time
would not be desirable. Remember, Klenow is the fragment of DNA pol I that
lacks the 5' to 3' nuclease activity, and was originally generated (and still
sold by some companies) as a purified proteolytic fragment. Even with cloned
Klenow the possibility still exists for other nucleases to co-purify with the
In addition to the possibility of contaminating nucleases, Klenow still retains
3' to 5' proofreading nuclease activity. Prolonged exposure of DNA to high
amounts of Klenow can result in 3' recessed ends due to this activity, thus
you may lose some of your labeled probe by increasing the length of incubation.
The original random-primed protocol of Feinberg and Vogelstein used random
hexamers for priming. This necessitated the use of low incubation temperatures
to allow annealing of the hexamers to the denatured DNA fragment. And, at the
lower temperature (room temp), polymerization is not very efficient. With the
advent of random octamers/nonamers/decamers the incubation temperature can be
raised to 37 C, and polymerization is very rapid.
This brings me to the second part: Klenow was originally isolated as the heat
stable fragment of E. coli pol I. Thus, it is incorrect to say that Klenow is
unstable. Yes, the reaction for all intensive purposes is over quickly at 37 C
(depending on the amount of starting DNA), but it is not due to lack of enzyme
activity, but rather the efficiency of polymerization at 37 C.
I hope this helps.
Dept. of Virology and Molecular Biology Internet: neale at mbcf.stjude.org
St. Jude Children's Research Hospital Phone: (901) 522-0400
Memphis, TN Fax: (901) 523-2622
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