Reports

Optimization of sample preparation - filtration and DNA extraction - for the analysis of sea water samples

Published:

01/11/2010

Authors:

Eyjólfur Reynisson, Árni Rafn Rúnarsson, Sveinn Haukur Magnússon, Desiree Seehafer, Viggó Þór Marteinsson

Supported by:

Fisheries Project Fund, Ministry of Fisheries and Agriculture

Contact

Viggó Marteinsson

Research Group Leader

viggo.th.marteinsson@matis.is

Optimization of sample preparation - filtration and DNA extraction - for the analysis of sea water samples

Little is known about microorganisms or the diversity of microbial communities in Icelandic waters, but they play an important role in the marine ecosystem. It is necessary to study the microbiology of the ocean around Iceland with new and powerful methods based on molecular biology. In such work, the quality of the samples and sample preparation are very important. In this study, a preliminary survey of sea samples, sampling and sample handling was performed before large quantities of samples are taken. First, samples were taken from the marina in Reykjavík for preliminary study and then we continued with samples from the open sea. Yields were examined for DNA levels and how well the microorganisms' genes were amplified by PCR. The results showed that the best method was a purchased DNA isolation kit that isolated most of the DNA and was quantifiable by PCR. A cheaper and faster method with an automatic isolator and home-made substrates also proved to be very successful, as comparable results were obtained from PCR amplification, although lower DNA recovery was obtained. Based on these results, it is possible to set up procedures based on automatic DNA isolation of samples but the use of purchased isolation kits on more difficult samples. It is planned to use these results for sea samples from the Marine Research Institute's spring survey.

The knowledge on microbial diversity and community structure in Icelandic seawater is scarce at present despite their important role in ocean ecology. The agenda is to increase our knowledge in this field by applying recent and powerful analytical tools. In order to do that it is essential to have access to high quality samples and sample preparation procedures. In the present study sea sample preparation was studied with aim of comparing different methods and optimizes the workflow. Samples from a harbor in Reykjavík and open sea samples were used for this purpose. The results showed that an extraction method based on an Epicenter kit gave the best results regarding DNA recovery from the samples and suitability in a PCR amplification. However, a method based on semi ‐ automatic protocol and in house reagents proved to be more cost effective and showed comparable performance with PCR suitability of the samples although a lower DNA recovery was obtained. From these results it is now possible to establish an efficient work flow for microbial diversity analysis of sea samples using an automated method as a first choice with the option of more costly method for more challenging samples.

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