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

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.


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