I used to study DNA repair of UV damage for a living. Let me share with
you what I know.
The short answer is UV destroys DNA very well as a sterilization technique
in most situations. 254 nm light is optimal.
The long answer:
A germicidal lamp in a hood puts out about 1 Joule/meter square/sec at the
hood surface. This is equivalent to a 17 inch 15 W germicidal lamp at 45
cm. In the absence of UV shielding by things such as dNTP, and assuming
better than 50% transmittance of 254 nm UV light in the sample, you can put
an average of one UV pyrimidine dimer in a 4,000 bp plasmid in one minute.
You can scale this factor with important parameters such as your target
length, and the number of molecules you need to knock out. At 10 cm from
the light, the UV dose is about 3 to 4 times higher only. Clearly, for
those who contemplate 40 cycles of PCR, one molecule of undamaged
contamination is too much. The optimal wavelength is 254 nm because
pyrimidine dimers form near that wavelength, yet the main UV band of a
mercury lamp is at 254 nm. Pymidine dimers absorb UV light of various
wavelengths and undergo reversal to regenerate the pyrimidine monomers. At
higher wavelengths such as 310 nm, the photoreversal of UV dimers to native
DNA is substantial. Other than pyrimidine dimers, other adducts also form
at lower frequency. Longer UV irradiation will bring on the assistance of
those DNA and DNA-protein adducts.
An important consideration is UV equilibrium. Pyrimidine dimers form and
reverses even in 254 nm light such that at equilibrium, only 7% of any
possible site can form a dimer. As a result, if you do not have enough
sites per PCR target sequence, and if you have high number of targets and
too many PCR cycles, UV irradiation may not sterilize your sample.
The second consideration is the choice of DNA polymerase for PCR. Taq is
pretty sloppy, and will go through UV pyrimidine dimers at considerable
frequency. You can be more safe by allowing multiple UV damages to occur on
each template. A lower Km polymerase may allow less error-prone DNA
replication to occur. I have no first hand knowledge on this latter point.
A third consideration is to make sure you have multiple runs of several
pyrimidines (3 or 4 is good) in your target sequence, or UV dimer formation
would not be that substantial.
With dried DNA, the numbers are less solid, and the photoadducts are
sometimes different. Little is known about the photoequilibrium of such
adducts.
As a result of these limitations on situations where UV sterilization is
incomplete, John Hearst and his associates promoted a 365 nm UV plus
psoralen (4, 5', 8- trimethyl psoralen) approach to put psoralen adducts in
the DNA. Such adducts are strong stoppers of DNA replication. The only
problem is that it would not work easily on dried DNA. It works best as a
post-PCR sterilization before the tube is opened. I have studied psoralen
repair for years. It is user friendly.
This must be more than you wanted to know. Hope it is useful to somebody.
Tony
At 02:23 PM 7/21/98 -0700, Andy Ament wrote:
> <subject> 7/21/98
>Hello All,
Can anyone help me with this question. How well does UV light destroy DNA
for sterilization techniques and what wavelength is optimal? thanks!
>Andrew
>
>
>>
>>Received: from postal.incyte.com (postal [10.1.0.6]) by genomesystems.com
>(8.8.4/8.8.4) with ESMTP id LAA10678 for <ament@genomesystems.com>; Tue,
21 >Jul 1998 11:57:21 -0500 (CDT)
>>Received: from post.aecom.yu.edu (post.aecom.yu.edu [129.98.1.4])
>> by postal.incyte.com (8.8.8/8.8.8) with ESMTP id JAA10868
>> for <ament@genomesystems.com>; Tue, 21 Jul 1998 09:56:31 -0700 (PDT)
>>Received: by post.aecom.yu.edu (8.9.1/8.9.1) id IAA10769
>> for abrf-out; Tue, 21 Jul 1998 08:23:36 -0400 (EDT)
>>Received: from helix.promega.com (de.promega.com [198.150.28.10])
>> by post.aecom.yu.edu (8.9.1/8.9.1) with ESMTP id IAA10764
>> for <abrf@aecom.yu.edu>; Tue, 21 Jul 1998 08:23:35 -0400 (EDT)
>>Received: from msn-ex1.promega.com (msn-ex1.promega.com [198.150.30.11])
by >helix.promega.com (8.8.8/8.8.8) with ESMTP id IAA20092 for
<abrf@aecom.yu.edu>; >Tue, 21 Jul 1998 08:21:26 -0400 (EDT)
>>From: TFrancis@promega.com
>>Received: by msn-ex1.promega.com with Internet Mail Service (5.5.1960.3)
>> id <M0325RZK>; Tue, 21 Jul 1998 07:14:35 -0500
>>Message-ID: ><7D732AE626BBD011B29000805FFE49CB02237467@msn-ex1.promega.com>
>>Old-To: abrf@aecom.yu.edu
>>Subject: Gilson products
>>Date: Tue, 21 Jul 1998 07:14:34 -0500
>>MIME-Version: 1.0
>>X-Mailer: Internet Mail Service (5.5.1960.3)
>>Content-Type: text/plain
>>To: Recipients of ABRF List <abrf@aecom.yu.edu>
>>Sender: Association of Biomolecular Resource Facilities
><abrf-request@aecom.yu.edu>
>>Precedence: bulk
>>Errors-To: abrf-request@aecom.yu.edu
>>X-UIDL: 2780d026824231725294d1ebb7833b53
>>
>
>
>
************************************
Dr. Anthony T. Yeung, Ph.D.
Director, Fannie E. Rippel Biotechnology Facility
Member, Institute for Cancer Research
Fox Chase Cancer Center
7701 Burholme Ave. Philadelphia, PA 19111
Voice: 215-728-2488
FAX: 215-728-3647
email: AT_Yeung@FCCC.edu
http://www.fccc.edu/research/labs/yeung/
************************************