3800-3801
Nucleic Acids Research, 1995, Vol. 23, No. 18
© 1995 Oxford University Press
A method for preparation of fecal DNA suitable for
PCR
Rainer Deuter, Stephan Pietsch1, Sven Hertel1 and Oliver Muller*
Arbeitsgruppe Tumorgenetik, Max-Planck-lnstitut fur Molekulare Physiologie, D-44139 Dortmund, Germany and
1
Stadtische Kliniken, Chirurgische Klinik, D-44137 Dortmund, Germany
Received May 16, 1995; Revised and Accepted August 7, 1995
The analysis of nuclear DNA extracted from fecal specimens is
an important tool in different areas of molecular genetic research
reaching from cancer diagnostics (1-3) to population genetic
studies (4). Here we describe a short and simple method for the
purification of chromosomal DNA that is ready to be used in
PCR. Without further subcloning the resulting PCR products
can be used directly for heteroduplex analysis, sequencing or
other DNA analysing methods.
Very often it is difficult to obtain chromosomal DNA from
feces that is suitable for further analysis. Commonly two
problems are encountered. First the DNA tends to degrade during
longer periods of storage, secondly the DNA is not suitable for
enzymatic manipulation such as PCR. Both problems can be
traced to the presence of copurified excremental substances like
bilirubin and bile salts (5). Therefore substantial reduction of
these substances from the purified DNA is essential (6). Several
dietary materials are known to decrease the concentration of fecal
bile acids and to bind bile acids in vitro (5,7,8). We tested different
adsorption matrices to remove bile acids during DNA extraction:
immobilized BSA, cellulose, potato starch (all from Sigma,
Munich, Germany) and potatoflour(Honig, Postbus 45,1540 AA
Koog a/d Zaan, Netherlands) which is mainly an insoluble
mixture of cellulose, starch, lipids and salts.
Human stool specimens were collected, frozen and stored at
-80°C. 200 mg stool was homogenized in 600 JJ.1 stool lysis
buffer (SLP: 500 mM Tris-HCl pH 9.0, 50 mM EDTA, 10 mM
NaCl). To one fourth of the homogenate, 200 (J.1 SLP containing
100 mg of the adsorption matrix was added. The suspension was
mixed vigorously and centrifuged twice at 5000 and 13 000 g
each for 5 min to precipitate bacteria and other debris. After
digesting the clear supernatant with proteinase K at a concentration of 2.5 mg/ml the DNA was purified using a DNA spin
column recommended for DNA purification from blood and
tissue (Qiagen, Hilden, Germany). Column loading and washing
steps were performed as described by the manufacturer. The
DNA was eluted from the spin column in a final volume of 150
(j.1 distilled water and stored at -20°C until further use. The yield
of total DNA was determined by measuring the absorption at 260
nm. All preparations gave similar yields of 15-20 u.g DNA. At
first sight the use of different adsorption materials appeared to
have no influence on the yield or the quality of the extracted DNA
as judged by agarose gel electrophoresis (Fig. 1). However after
storing the DNA at -20°C all the preparations showed degrada-
* To whom correspondence should be addressed
M 1 2
3 4
Figure 1. Chromosomal DNA extracted from a single stool specimen in
absence (lane 1) of any adsorption matrix, in presence of BSA (lane 2), cellulose
(lane 3), potato starch (lane 4), or potato flour (lane 5). Lane M, DNA size
marker (lambda DNA digested with HindTO). One thirtieth part of the extracted
DNA was loaded onto a 0.7% agarose gel.
tion of DNA. DNA loss varied between different samples and
reached up to 80%. Compared with untreated preparations
preadsorption decreased the loss by degradation. The most stable
DNA preparations were obtained by using potato flour as an
adsorption matrix (Table 1).
For PCR 3 (il of the purified chromosomal DNA was used in
a total volume of 50 ul containing 10 mM Tris-HCl pH 8.3, 50
mM KC1,1.5 mM MgCl2,30 jiM each of dATP, dCTP, dGTP and
dTTP, 400 nM of each primer, 100 (ig/ml BSA and 0.75 U Taq
polymerase (AGS, Heidelberg). To increase sensitivity of the
PCR we routinely performed nested PCR methods as described
recently (9) using biotin labelled nested primers. PCR of DNA
samples purified in absence of any adsorption matrix was
completely inhibited (Fig. 2A). Only some of the DNA preparations purified in presence of BSA, cellulose or potato starch
showed reproducible PCR results. This was probably due to
varying concentrations of PCR inhibiting substances in the
different DNA samples (Table 1). Only when template DNA was
purified in the presence of potato flour reliable PCR results from
all analysed specimens were obtained (Fig. 2B). The PCR
fragments were successfully used for heteroduplex analysis and
Nucleic Acids Research, 1995, Vol. 23, No. 18 3801
Table 1. Properties of nuclear DNA extracted from stool
Matrix
Loss a (%)
None
80
PCRb
0
BSA
60
3
Cellulose
60
4
Potato starch
60
4
Potato flour
<5
10
a
DNA loss by degradation was measured after storage for one week at -20°C
by analytical agarose gel electrophoresis and spectrophotometric analysis (not
shown).
b
PCR of DNA samples from 10 different stool specimens was performed.
Number of samples which gave the expected PCR product are shown.
ACKNOWLEDGEMENTS
We are indebted to Alfred Wittinghofer and Dietrich Lohlein for
continous support.
REFERENCES
Figure 2. PCR results. Lane M, DNA size marker (1 kb DNA ladder). Nested
PCR was performed with primers specific for a gene region of the
Adenomatous Polyposis Coli gene. One tenth of the total PCR volume was
loaded onto 1.4% agarose gels. The identity of the amplified fragments was
proven by sequencing. (A) PCR results using template DNA extracted from the
same stool specimen in absence of any adsorption matrix (lane 1), in presence
of BSA (lane 2), cellulose (lane 3), potato starch (lane 4), or potato flour (lane
5). (B) Results of the PCR using template DNA extracted from three different
stool specimens in absence of any adsorption matrix (-) or in presence of potato
flour (+).
for direct sequencing. For direct sequencing single strand DNA
preparation was performed using streptavidin coupled magnetic
beads and following the instructions of the manufacturer (Dynal,
Hamburg, Germany).
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