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The Use of MasterTaq Polymerase for PCR with Humic Material-Contaminated ,,, DNA

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

  1. Steffan RJ, Goksoyr J, Bej AK, and Atlas RM. Recovery of DNA from soils and sediments. Appl Environ Microbiol. 1988;54:2908-2915.
  2. 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.
  3. 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;5
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