Methods and reagents: Determination DNA concentrations and rescuing PCR primers
by Paul N. Hengen, Ph.D. *
Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
Internet. This month's column reports on the relative merits of methods
for determining DNA concentrations, and how to rescue contaminated polymerase
chain reaction (PCR) primers.
Determination of nucleic acid concentrations
Accurately determining the DNA concentrations of crude chromosomal or
purified plasmid DNA samples is not only desirable, but an essential step
in quantitative manipulations of DNA. Researchers often find it difficult
to interpret the results of spectrophotometric and/or fluorometric analyses,
so they look for alternative methods.
One netter was understandably confused by a discrepancy in measurements using
different techniques for estimating the concentration of a dilute sample of
plasmid DNA that was purified by two passes through an ethidium bromide -
cesium chloride (EtBr-CsCl) centrifugation gradient.
The sample was tested on an LKB Biochrom Ultrospec II spectrophotometer for
absorbance at wavelengths of 260 nm and 280 nm, as well as for emission of 460
nm on the Hoefer TKO 100 mini-fluorometer in the presence of bisbenzimidizole,
a fluorescent dye known as Hoechst H 33258 (manufactured by American Hoechst
Corporation), that has an excitation maximum at 356 nm and an emission maximum
of 458 when bound to DNA [1]. The A260:A280 ratio for the sample, using the
spectrophotometer, was 1.80, indicating highly purifed DNA free from
contaminating protein. However, there was a threefold lower estimation of DNA
using the fluorometer.
The reason for this discrepancy could be that the spectrophotometer detects
absorbance due to RNA as well as DNA, while the Hoescht dye used in the
fluorometer interacts specifically with adenosine and thymidine residues of
DNA. Some netters argued that an RNase treatment would have eliminated all RNA,
and that the spectrophotometer reading was more trustworthy, while others were
more willing to believe the fluorometer value because it was possible that
contaminating RNA polynucleotides of similar buoyant density banded with the
covalently closed circular plasmid DNA, causing a higher absorbance reading.
During an EtBr-CsCl centrifugation, some remaining RNA might also adhere to the
side of the ultracentrifuge tube and could be withdrawn into a syringe along
with the banded DNA.
On the other hand, the unexpectedly low value from the fluorometric assay could
have been due to some EtBr remaining in the DNA sample, causing displacement of
the Hoechst dye during the assay. The lower reading could also be due to a
deviation from the optimum buffer pH of 7.4, a critical parameter for zeroing
the emission of Hoechst H 33258 at 458nm. This would produce higher background
emission levels from the dye alone, hence lower estimates of DNA
concentration.
One person experienced with the mini-fluorometer wrote that it is accurate for
quantitation of crude chromosomal DNA, but not reliable for plasmids and other
DNA of limited complexity. This could be due to the highly specific nature of
the Hoechst dye, so that it might be necessary to perform standardizations for
individual DNA molecules of differing AT content. It could also be accounted
for by reduction of the background signal due to competition for DNA between
the Hoechst dye and DNA binding proteins within cell extracts, or perhaps the
correct DNA conformation is needed to enhance fluorescence over background
emission.
Others found the fluorometer to be faster, easier and more accurate than a
spectrophotometer for all DNA samples, since the response of the interactive
dye is nearly linear over a 1000-fold range. It is only necessary to make a
single standard adjustment to estimate DNA concentrations between five and
2000 ng/ul. In addition, the mini-fluorometer can be fitted with an adaptor
to accept disposable capillary tubes for measurements of 2-10 ul of solution,
allowing the recovery of the DNA.
Alternative methods
Other methods for quantitating DNA may be even faster. For example, Invitrogen
sells a nucleic acid quantitation DNA Dipstick [TM] kit, which is claimed to be
sensitive enough to detect as little as 0.1 ng/ul of nucleic acid, and the
entire procedure is advertised to take only 10 minutes. Disadvantages include
the fact that it is difficult to process multiple samples simultaneously, and
impossible to determine the purity of the sample, as can be done using a
spectrophotometer. Unfortunately, the method cannot be used with samples
containing more than 10 ng/ul of nucleic acids, requiring a series of dilutions
for more concentrated solutions. Also, in practice, each test takes nearly
30 minutes. (For a general discussion on the use of kits, see TIBS 19, 46-47.)
A simple and very inexpensive method discussed is the use of EtBr agarose
plates. DNA samples of 2-10 ul are spotted onto 1% agarose containing 0.5
ug/ml EtBr within a Petri dish. Afterwards, the plate is exposed to UV light
and photographed. Another variation is to mix 5-10 ul of a 0.5 ug/ml solution
of EtBr with 10 ul of DNA spotted onto plastic film wrap or a siliconized glass
slide placed on top of a UV transilluminator. The advantage of this method is
that DNA samples with as little as 1-10 ng of DNA can be quantitated within
minutes. The disadvantage is the intercalation of the dye with RNA as well as
DNA.
While the method for determining the concentration of a DNA sample most favored
by purists is by using spectrophotometry, for quick results, the EtBr spot test
is the method of choice among nearly all users on methds-reagnts.
Rescuing contaminated PCR Primers
Michael Lush
(mjl17@mbuc.bio.cam.ac.uk)
suspected that he had accidentally
contaminated a PCR primer stock solution with a DNA fragment of approximately 6
kb in length. Since it would be very expensive to replace the entire primer
solution, he asked for advice on how to eliminate the contaminating DNA
fragment.
Provided the proper equipment and experienced personnel are available, the most
reliable method would be to use strong-anion-exchange chromatography or
reverse-phase chromatography. One person suggested purifying the primer stock
on a polyacrylamide gel and extracting the banded oligonucleotide. However,
any of these would be time consuming.
One faster and less expensive alternative for separating the primers from the
double-stranded (ds) DNA fragments would be to spin the mixture through a
Millipore Ultra-free [TM]-MC filter unit containing a membrane with a 30 kDa
cut-off, or a Centricon 100 column (Amicon Inc.), which has a 100 kDa cut-off.
This would trap the larger DNA molecules onto the membrane, while allowing the
smaller primers to pass through into the collection reservoir. Using this
purification scheme, however, might not remove all the contaminating DNA since
it could have been partially degraded, and smaller fragments might pass through
the membrane and end up in the filtrate.
Another suggestion was to place the solution in a clear microcentrifuge tube
and expose it to 254 nm UV light for a minimum of five minutes. Presumably,
this would crosslink the larger DNA fragments and prevent annealing of the
primer, or prohibit the polymerase from passing the crosslinked region during
extention. For example, ten minutes on a Statalinker UV crosslinker from
Statagene should be sufficient to eliminate visible bands of amplified template
DNA during PCR [2].
Alternatively, the primers could be treated by adding at least 0.5 units of
DNase to the concentrated stock solution or to the PCR mix before adding the
template and polymerase. Incubation for ten minutes at room temperature,
followed by boiling for ten minutes, would probably eliminate the unwanted
dsDNA and also destroy the DNase activity.
Although it might not be necessary for every application, some people use DNase
religiously to treat PCR reactions before template addition, regardless of
suspected contamination. This might not always be a good idea, and care must
be taken to remove all remaining DNase, as even the slightest residual activity
could be detrimental to the amplification of low-abundance DNA.
A problem that DNase treatment has in common with spin filtration is that
partial digestion could allow small fragments of single-stranded DNA left in
the solution to act as primers, leading to possible false positive bands or
smears of multiple PCR products seen after agarose gel electrophoresis of the
sample.
Using a combination of DNase, UV light and filtration might be sufficient to
allow the use of the primers for routine PCR applications. Although loss of
the primer stock would be costly, most netters felt that the risk of obtaining
extraneous PCR products from the suspect primer stock would be too great and
that, once contaminated, the primers would not be sufficiently clean to be
trusted.
References
[1] Labarca, C., and Paigen, K. (1980) Anal. Biochem. 102, 344-352
[2] Sarker, G., and Sommer, S. S. (1990) Nature 343, 27
Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health.
Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author.
You found this at the World Wide Web (WWW) Uniform Resource Locator (URL):
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/feb94.txt
Any reference to this column must be cited as the following published
article:
@article{Hengen1994Febtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Determining {DNA} concentrations
and rescuing {PCR} primers",
journal = "Trends in Biochemical Sciences",
volume = "19",
number = "2",
pages = "93-94",
month = "February",
year = "1994"}
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******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - - Methods FAQ list -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - - * * - TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - - * * - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/ * ******************************************************************************* |
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