PROTOCOL

Isolation of DNA from bacterial samples of the human

gastrointestinal tract
Erwin G Zoetendal1,2, Hans GHJ Heilig1, Eline S Klaassens1, Carien CGM Booijink1,2,
Michiel Kleerebezem2,3, Hauke Smidt1,2 & Willem M de Vos1,2
1Laboratory

of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT, Wageningen, The Netherlands. 2Wageningen Centre for Food Sciences,
Diedenweg 20, 6703 GA, Wageningen, The Netherlands. 3NIZO Food Research B.V., Kernhemseweg 2, 6718 ZB, Ede, The Netherlands. Correspondence should be
addressed to W.M.dV. (Willem.devos@wur.nl)

2006 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 27 July 2006; doi:10.1038/nprot.2006.142

The human gastrointestinal (GI) tract contains a complex microbial community that develops in time and space. The most widely used
approaches to study microbial diversity and activity are all based on the analysis of nucleic acids, DNA, rRNA and mRNA. Here, we
present a DNA isolation protocol that is suitable for a wide variety of GI tract samples, including biopsies with minute amounts of
material. The protocol is set up in such a way that sampling can be performed outside the laboratory, which offers possibilities for
implementation in large intervention studies. The DNA isolation is based on mechanical disruption, followed by isolation of nucleic
acids using phenol:chloroform:isoamylalcohol extraction. In addition, it includes an alternative DNA isolation protocol that is based
on a commercial kit. These protocols have all been successfully used in our laboratory, resulting in isolation of DNA of sufficient
quality for microbial diversity studies. Depending on the number of samples and sample type, the whole procedure will take
approximately 2.54 hours.

INTRODUCTION
Microbial ecosystems are found all over the world, ranging from the
bottom of the ocean to the stratosphere. Without these microbial
ecosystems, life on earth would not be possible as they are involved
in recycling the elements that are crucial for all life forms. Our
gastrointestinal (GI) tract is inhabited by large numbers of
microbes, which collectively outnumber host cells by a factor of
10 (ref. 1). The main function of the GI tract is the conversion of
food into easily absorbable and digestible components: the microbial community converts the indigestible components into nutrients that can be taken up by the epithelium. The microbial
community in the GI tract consists of different groups of microbes,
of which anaerobic bacteria are the most numerous. This community structure is host-specific and affected by the genotyope, and its
diversity varies in time and in space, which makes the GI tract a
highly complex ecosystem to study2. Currently, the estimated
number of bacterial species in the human GI tract is more than
1,000. It is generally accepted that we cannot cultivate all microbes
from the human GI tract and, therefore, culture-independent
nucleic-acid-based approaches have been introduced to study
microbial diversity and activity. Most of these approaches focus
on the use of 16S rRNA and its corresponding gene because 16S
rRNA is present in every cell, has a low mutation and horizontal
transfer rate, and more than 200,000 16S rRNA (gene) sequences
are available, at present, in various databases3. Therefore, it has
been used as a phylogenetic marker for the detection, identification
and quantification of uncultured microbes from a variety of
ecosystems, including the human GI tract. The first and most
critical step in the 16S rRNA approach is a reliable isolation of
nucleic acids from GI tract samples, as the quality of all subsequent
procedures are dependent on this step. In this paper, we will
describe the isolation of DNA from intestinal samples. The isolation of RNA will be described elsewhere4.
An extra complication with studying the human GI tract is the
fact that intestinal samples cannot always be processed freshly, as
870 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS

sampling often occurs at the homes of volunteers or in hospitals

especially when sampling concerns babies; volunteers without
a colon, such as ileostomy patients; or biopsy specimens from
routine clinical procedures. This argues for proper preparation
and storing methodologies that are easy to implement. In our
laboratory, we have developed and validated protocols for the
isolation of DNA from intestinal samples, which are then ready
to use for PCR-based diversity analysis. These protocols have
been used successfully for feces57, ileostomy effluent and tissue
samples8, and the DNA has served as a target for 16S rRNA
gene-based approaches. In this paper, we present a protocol for
the isolation of DNA from intestinal samples. The first part
of the protocol, which is based on mechanical disruption
followed by isolation of DNA using phenol:chloroform:isoamylalcohol extraction, has been adapted from Zoetendal et al.9. The
second (alternative) part of the DNA isolation protocol that
is described is adapted from a commercial kit, Isolation of DNA
from Stool for Pathogen Detection (Qiagen). Both parts of
the protocol have been successfully used in our laboratory,
resulting in the isolation of DNA of sufficient quality for
microbial diversity studies. We believe that the use of this
protocol is not limited to samples from the human GI tract,
but that it can also be applied to samples from other microbial
ecosystems.
Procedural comments
The procedure for DNA isolation depends on the type of sample
(feces, ileostomy effluent or biopsy specimen). Therefore, the DNA
isolation protocol consists basically of two steps, which includes
processing of the sample followed by one of the DNA isolations
provided.
Wearing gloves during DNA isolation is highly recommended.
This minimizes contamination of samples and it protects you
against the adverse effects of toxic or harmful reagents.

PROTOCOL
MATERIALS
REAGENTS

. Acetic acid (glacial) (cat. no. 27225; Riedel-de Haen) ! CAUTION Corrosive.
Handle using appropriate safety equipment.

. ASL buffer (included in cat. no. 51504; Qiagen Inc.)

. Buffer-saturated phenol (cat. no. 15513-039; Invitrogen) ! CAUTION Phenol
is toxic. Handle using appropriate safety equipment and measures.

. Chloroform (cat. no. 24216; Riedel-de Haen) ! CAUTION Harmful. Handle

using appropriate safety equipment.

. EDTA (cat. no. E5134; Sigma)

. 95% ethanol (cat. no. 20 824.365; VWR BDH Prolabo) ! CAUTION Ethanol
is flammable. Handle using appropriate safety equipment and measures.

. Glycogen, 5 mg ml1 (cat. no. 9510; Ambion)

. HCl (cat. no. 30721; Riedel-de Haen) ! CAUTION Corrosive. Handle using
2006 Nature Publishing Group http://www.nature.com/natureprotocols

appropriate safety equipment.

. Isoamylalcohol (cat. no. 59085; Riedel-de Haen) ! CAUTION Harmful.

Handle using appropriate safety equipment.

. KCl (cat. no. 31248; Riedel-de Haen)

. KH2PO4 (cat. no. 30407; Riedel-de Haen)
. NaAc (cat. no. S7545; Sigma)
. NaCl (cat. no. 31434; Riedel-de Haen)
. Na2HPO4 . 2H2O (cat. no. 04272; Riedel-de Haen)
. Proteinase K, 20 mg ml1 (cat. no. p2308; Sigma)
. SDS (cat. no. L4390; Sigma) ! CAUTION Harmful. Handle using appropriate
safety equipment.

. Tris (cat. no. T1378; Sigma)

EQUIPMENT

. Screw-cap tubes (cat. no. 171072 and 172009; BIOplastics BV)

. Oak Ridge centrifuge tubes (cat. no. 3119; Nalgene Labware)
. Corning Lambda single channel pipettors (cat. no. 49584964; Corning
Inc.)

. Corning pipette tips (cat. no. 4808 and 4810; Corning Inc.)
. Eppendorf centrifuge (cat. no. 5415R; Eppendorf)

. Eppendorf Thermomixer compact (Eppendorf)

. FastPreps FP220A (cat. no. 6001-220; MP Biomedicals)
. Glass beads, 3 mm (cat. no. 290004; Omnilabo Int.)
. Gloves, TT Clear (Maxxim Medical Europe)
. Isolation of DNA from Stool for Pathogen Detection (cat. no. 51504;
Qiagen Inc.)

. Microfuge tubes, 1.5 ml (cat. no. 04-210-1100; Nerbe Plus)

. Microfuge tubes, 2.0 ml (cat. no. 623 201; Greiner Bio)
. MS2 Mini Shaker ST0001 (Appleton Woods)
. Tissue paper Kimcare medical wipes (Kimberly-Clark)
. Zirconia beads (cat. no. 11079101Z; Biospec products)
REAGENT SETUP
Chloroform:isoamylalcohol Mix chloroform and isoamylalcohol in a 24:1
volume ratio. ! CAUTION Both chloroform and isoamylalcohol are harmful.
Handle using appropriate safety equipment and measures.
Ethanol 70% Mix 95% ethanol and milliQ water in a 7:3 volume ratio and
store at 20 1C. ! CAUTION Ethanol is flammable. Handle using appropriate
safety equipment and measures.
3 M NaAc Dissolve 408.1 g of NaAc . 3H2O in 800 ml milliQ water. Adjust pH
to 5.2 with glacial acetic acid. Add milliQ water until the total volume is 1.0 L.
13 phosphate-buffered saline (PBS) Dissolve, per 1.0 L milliQ water: 8.0 g
NaCl, 0.2 g KCl, 1.44 g Na2HPO4 . 2H2O and 0.24 g KH2PO4. Adjust to pH 7.4.
10% SDS Dissolve 100 g SDS in 800 ml milliQ water. Heat to 68 1C to
enhance the dissolving process of SDS. Adjust to pH 7.4. Add milliQ water
until the total volume reaches 1.0 L. ! CAUTION SDS is harmful. Handle using
appropriate safety equipment and measures.
TE buffer Make a solution consisting of 10 mM Tris-HCl (pH 7.6) and 1 mM
EDTA (pH 8.0).
Tris-HCl Dissolve 121.1 g Tris in 800 ml milliQ water. Adjust to pH 7.6 with
HCl. Add milliQ water until a volume of 1.0 L is reached. Use this solution for
the preparation of all Tris-HCl-based solutions. ! CAUTION HCl is corrosive.
Handle using appropriate safety equipment and measures.

PROCEDURE
Sample preparation for DNA isolation
1| The sample preparation is dependent on the type of intestinal sample; procedures are given for sample preparation from
feces (A), ileostomy effluent (B) and biopsy (C) specimens.
(A) Sample preparation from feces
(i) Weigh approximately 0.2 g of feces.
PAUSE POINT Samples can be stored at 4 1C for 1 day or at 20 1C for several weeks.
m CRITICAL STEP Although samples can be stored for a long time at 20 1C, we recommend processing the samples as quickly
as possible. Longer storage may result in differential lysis of microbial cells.
(ii) Add sample into a microfuge tube.
(iii) Resuspend sample in 1 ml TE or 1.4 ml ASL buffer.
(iv) Continue with Step 2A (when TE buffer is added) or Step 2B (when ASL buffer is added).
(B) Sample preparation from ileostomy effluent
(i) Weigh approximately 510 g of ileostomy effluent sample in a centrifuge tube.
PAUSE POINT Samples can be stored at 4 1C for 1 day or at 20 1C for several weeks.
m CRITICAL STEP Although samples can be stored for a long time at 20 1C, we recommend processing the samples as quickly
as possible. Longer storage may result in differential lysis of microbial cells.
(ii) Add 10 ml 1 PBS and 10 glass beads (3 mm).
(iii) Vortex for 30 s.
m CRITICAL STEP This step is performed to separate cells from debris. Sometimes additional mechanical shearing
by pipetting may enhance recovery of the cells.
(iv) Centrifuge at 300g at 4 1C for 5 min.
(v) Transfer supernatant and centrifuge at 9,000g at 4 1C for 10 min.
(vi) Remove supernatant and add 1 ml of TE or 1.4 ml ASL buffer.
(vii) Continue with Step 2A (when TE buffer is added) or 2B (when ASL buffer is added).
(C) Sample preparation from biopsy specimens
(i) Add biopsy specimen to 1 ml TE buffer.
PAUSE POINT Samples can be stored at 4 1C for 1 day or at 20 1C for several weeks.
NATURE PROTOCOLS | VOL.1 NO.2 | 2006 | 871

PROTOCOL

2006 Nature Publishing Group http://www.nature.com/natureprotocols

m CRITICAL STEP Although samples can be stored for a long time at 20 1C, we recommend processing the samples as quickly
as possible. Longer storage may result in differential lysis of microbial cells.
(ii) Add 50 ml 10% SDS and 10 ml Proteinase K (20 mg ml1) to 1 ml samples.
! CAUTION SDS is harmful. Handle using appropriate safety equipment and measures.
(iii) Incubate at 55 1C in an Eppendorf Thermomixer for 1 h.
(iv) Continue with Step 2A.
m CRITICAL STEP We have not validated the DNA isolation described in Step 2B for human biopsy specimens and,
therefore, we cannot guarantee that this procedure will be successful.
2| Two procedures for DNA isolation are described below, the second of which is adapted from the protocol Isolation of DNA
from Stool for Pathogen Detection (Qiagen).
(A) Phenol:chloroform-based DNA isolation
(i) Pipette resuspended intestinal sample into a screw-cap tube containing 0.3 g zirconia beads.
(ii) Pipette 150 ml buffer-saturated phenol to the sample.
! CAUTION Phenol is toxic. Handle using appropriate safety equipment and measures.
(iii) Treat sample in the FastPrep at 5.5 ms 1 for 3 min.
(iv) Cool sample on ice for 1 min.
(v) Pipette 150 ml choroform:isoamylalcohol (24:1).
! CAUTION Both choloroform and isoamylalcohol are harmful. Handle using appropriate safety equipment and
measures.
(vi) Mix well and centrifuge at 15,700g at 4 1C for 5 min.
(vii) Transfer the upper layer into a new microfuge tube.
(viii) Pipette 150 ml phenol and 150 ml chloroform:isoamylalcohol.
(ix) Mix and centrifuge at 15,700g at 4 1C for 2 min.
(x) Transfer the upper layer into a new microfuge tube.
(xi) Repeat Steps 2A(viii) to 2A(x) until the interface of the two layers is clean.
(xii) Pipette 300 ml choroform:isoamylalcohol (24:1).
(xiii) Mix well and centrifuge at 15,700g at 4 1C for 5 min.
(xiv) Transfer the upper layer into a new microfuge tube.
(xv) Pipette 4 ml 5 mg ml1 glycogen and mix.
m CRITICAL STEP Glycogen will co-precipitate with the DNA in ethanol9. This step is necessary when DNA is isolated
from biopsy specimens or ileostomy effluent because DNA might be present at a low concentration. For DNA isolation
from feces, this step is not necessary.
(xvi) Pipette 1/10 volume of 3 M NaAc (pH 5.2) and 2 volumes of cold (20 1C) 95% ethanol.
! CAUTION Ethanol is flammable.
(xvii) Store at 20 1C for 30 min.
(xviii) Centrifuge at 15,700g at 4 1C for 20 min.
(xix) Remove supernatant, add 500 ml cold (20 1C) 70% ethanol and mix.
! CAUTION Ethanol is flammable.
(xx) Centrifuge at 15,700g at 4 1C for 5 min.
(xxi) Dry pellet by putting tube upside down on tissue paper for 15 min.
(xxii) Rehydrate pellet in 100 ml TE buffer (pH 8.0).
? TROUBLESHOOTING
(B) Qiagen kit-based DNA isolation
(i) Pipette the resuspended intestinal sample into a screw-cap tube and add 0.5 g 0.1 mm zirconia beads and 4 glass beads.
(ii) Vortex continuously until the sample is thoroughly homogenized.
(iii) Treat sample in a FastPrep at 5.5 ms 1 for 3  30 s, with cooling on ice between each treatment.
(iv) Heat at 95 1C in an Eppendorf thermomixer for 15 min.
(v) To continue and finish DNA isolation, proceed to Step 4 of the protocol Isolation of DNA from Stool for Pathogen
Detection (Qiagen).
? TROUBLESHOOTING

TIMING
DNA isolation from 12 GI tract samples can be performed in approximately 2.53 h.

872 | VOL.1 NO.2 | 2006 | NATURE PROTOCOLS

PROTOCOL
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.

le

Fe

TABLE 1 | Troubleshooting table.

PROBLEM
No DNA visible on gel
No DNA visible on gel
DNA not suitable for PCR

POSSIBLE REASON
DNA degradation took place
Low cell number in sample*
Impurities in template DNA

SOLUTION
Repeat DNA isolation
Pool DNA samples
Dilute DNA 10 or 100 timesw
or add BSA to PCR mix

2006 Nature Publishing Group http://www.nature.com/natureprotocols

*This may particularly be observed with ileostomy effluent and/or biopsy specimens.
wN.B. Diluting nucleic acids for PCR purposes may require additional amplification cycles. However, this may result in a more biased
representation of the initial diversity.
BSA, bovine serum albumin.

ANTICIPATED RESULTS
In our laboratory, these DNA isolation procedures have been used successfully for GI
tract samples. Typical concentrations of nucleic acids isolated from these samples are
listed in Table 2.

23.1
9.4
6.5

HMW DNA

4.3

2.3
2.0

0.6

Degraded NA

TABLE 2 | Range of nucleic acid concentrations obtained during the

isolation procedures.
Sample
Feces
Ileostomy effluent
Biopsy specimen

DNA (lg g1 sample)

1030*
26w
N.A.

*Data from Klaassens, unpublished results.

wData from Booijink, unpublished results.
N.A., not applicable, as biopsies contain only approximately 106 bacteria per mg and hence the majority
of the DNA from the biopsies is of human origin8.

Figure 1 | Agarose gel showing examples of DNA

isolated from feces (Fe) and ileostomy effluent
(Ie). M indicates the l HinDIII marker from which
the sizes of each fragment are indicated in kbp.
HMW DNA: high-molecular-weight DNA; degraded
NA, degraded nucleic acids.

The DNA often appears as a smear on agarose gels (Fig. 1), which is likely to be the result of mechanical disruption. Enzymatic
and chemical lysis procedures result in DNA of high molecular weight. However, their success is dependent on the cell wall
and membrane structures of the microbes, which are unknown for the majority of microorganisms. Therefore, we recommend
mechanical cell lysis for the isolation of nucleic acids from GI tract samples. The DNA can be used for all 16S-rRNA-gene-based
approaches, such as cloning of 16S rRNA genes, PCR-DGGE of 16S rRNA genes and real-time PCR. In addition, we have ample
experience that other genes can also be easily targeted.

COMPETING INTERESTS STATEMENT The authors declare that they have no

competing financial interests.
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
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