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Katrin Ravenschlag
Max-Planck-Institute for Marine Microbiology, Celsiusstr.1, 28359 Germany
Phone: ++49-421-2028-936, Fax: ++49-421-2028-580, email: kravensc@mpi-bremen.de
Introduction
Direct extraction of total DNA from soils or sediments always results in coextraction of other soil components, mainly humic acids or other humic substances, which negatively interfere with DNA detection and biochemical reactions.1 This contamination can, for example, inhibit Taq Polymerase in PCR*,25 or restriction endonucleases.6,3 Humic material, even in quantities as small as 1 ng, has been shown to inhibit PCR.7 Humic acids and nucleic acids share similar physico-chemical properties, making the complete removal of humic material difficult. Numerous studies on the development of an effective purification method have been conducted but none appears universal (see References 810). In this article, I shall be reporting on the excellent suitability of MasterTaq polymerase for the amplification of 16S rDNA genes from humic substances-contaminated DNA extracted from a marine sediment (Svalbard, Arctic Ocean).
Materials and Methods
DNA extraction. Total DNA was directly extracted from the sediment according to Zhou et al.10 The protocol encompassed three cycles of freezing and thawing, chemical lysis in a high-salt extraction buffer by heating the suspension in the presence of sodium dodecyl sulfate (SDS) and hexadecyltrimethylammonium bromide (CTAB), and a proteinase K step. The crude DNA was purified by dialysis. However, there were humic substances left in the DNA extract indicated by the brownish color of the DNA.
PCR amplification. Two universal bacterial primers EUB008 and
EUB1492 were used to amplify 16S rDNA genes from the extracted and purified
chromosomal DNA.11,12 PCR was performed with a Eppendorf Mastercycler
gradient** as follows: 50 pmol of each primer, 2.5 mol of each
deoxyribonucleoside triphosphate, 300 g bovine serum albumin (BSA),
1x reaction buffer, 1x TaqMaster PCR enhancer and 1U MasterTaq DNA
Polymerase were adjusted to a final volume of 100 l with sterile
water. From 80 ng to 500 ng of template DNA was added to the reaction
mixture after preheating to 70C to avoid nonspecific annealing of
the primers to nontarget DNA. The cycles used were:
1 cycle at 70C for 1 min, followed by 38 cycles at 95C for 1 min, 40C for 1 min, 72C for 3 min and 1 final cycle at 95C for 1 min, 40C for 1 min and at 72C for 10 min. In parallel, the same experiment was performed using a conventional Taq polymerase, which was routinely used in our lab and considered to be of maximal suitability for PCR. DNA extracted from E. coli was used as a positive control.
The use of high concentrations of BSA in the hot-start PCR was shown to decrease the sensitivity of the reaction against components in the soil extracts and to make PCR product formation reproducible.
Fig. 1: Use of MasterTaq and conventional Taq polymerase for the amplification
of 16S rDNA genes from humic-acid contaminated DNA extracted from a marine
sediment. PCR products were analyzed on a 1% agarose gel.
Lanes 1 and 4: E. coli-DNA (= positive controls)
Lanes 2 and 5: negative control (H2O)
Lanes 3 and 6: community DNA, extracted from a marine sediment
Fig. 2: Sampling of marine sediment with subsequent DNA preparation and PCR
Results and Discussion
The 16S rDNA gene from E. coli could be amplified successfully with both MasterTaq polymerase (Fig.1, lane 1) and a conventional Taq polymerase (lane 4). The use of MasterTaq polymerase for specific amplification of 16S rDNA of humic acid-contaminated DNA extracted from marine sediment resulted in a high yield of PCR product (lane 3). However, with a conventional Taq polymerase 16S rDNA amplification from the community DNA failed. Optimization of PCR conditions (amount of template DNA, number of cycles) did not result in a PCR product either.
PCR using another primer set (PCR product length 650 bp) and the same DNA extract always resulted in a higher yield of PCR product when using MasterTaq polymerase than when using a conventional Taq polymerase (data not shown). MasterTaq polymerase seems to be highly suitable for PCR with humic-rich DNA extracted from marine sediment. MasterTaq polymerase was not significantly inhibited by humic substances and seems to be very useful for problematic samples.
References
- Steffan RJ, Goksoyr J, Bej AK, and Atlas RM. Recovery of DNA from soils
and sediments. Appl Environ Microbiol. 1988;54:2908-2915.
- Smalla K, Cresswell N, Mendonca-Hagler LC, Wolters A, and van Elsas JD.
Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated
amplification. J Appl Bacteriol. 1993;74:78-85.
- Tebbe CC and Vahjen W. Interference of humic acids and DNA extracted directly
from soil in detection and transformation of recombinant DNA from bacteria
and a yeast. Appl Environ Microbiol. 1993;59(8):2657-2665.
- Tsai YL and Olson BH. Detection of low numbers of bacterial cells in soils
and sediments by polymerase chain reaction. Appl Environ Microbiol. 1992;58:754-757.
- Tsai YL and Olson BH. Rapid method for separation of bacterial DNA from
humic substances in sediments for polymerase chain reaction. Appl Environ
Microbiol. 1992; 58:2292-2295.
- Portous LA and Armstrong JL. Recovery of bulk DNA from soil by a rapid,
small-scale extraction method. Curr Microbiol. 1991;22:345-348.
Bej AK and Mahbubani MH. PCR Technology: Current Innovations. Boca Raton, Fl: CRC Press; 1994.
- Harry M, Gambier B, Bourezgui Y, and Garnier-Sillam E. Evaluation of purification
procedures for DNA extracted from organic rich samples: Interference with
humic substances. Analysis. 1999;27(5):439-442.
- Romanowski G, Lorenz MG, and Wackernagel W. Use of polymerase chain reaction
and electroporation of Escherichia coli to monitor the persistence of
extracellular plasmid DNA introduced into natural soils. Appl Environ
Microbiol. 1993;59:3438-3446.
- Zhou J, Brunns MA, and Tiedje JM. DNA recovery from soils of diverse composition.
Appl Environ Microbiol. 1996;62:316-322.
- Hicks RE, Amann RI, and Stahl DA. Dual staining of natural bacterioplankton
with 4, 6-diamidino-2- phenylindole and fluorescent oligonucleotide
probes targeting kingdom-level 16S rRNA sequences. Appl Environ Microbiol.
1992;68(7):2158-2163.
- Kane MD, Poulsen LK, and Stahl DA. Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences. Appl Environ Microbiol. 1993;59(3):682-686.
* Practice of the patented polymerase chain reaction (PCR) process requires a license.
** The Eppendorf Thermal Cycler is an Authorized Thermal Cycler and may be used with PCR licenses available from Applied Biosystems. Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Mastercycler gradient (U.S. Pat. 6,210,958)
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