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
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.