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A novel method for the measurement of dissolved deoxyribonucleic acid in seawater
Author(s) -
Brum Jennifer R.,
Steward Grieg F.,
Karl David M.
Publication year - 2004
Publication title -
limnology and oceanography: methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.898
H-Index - 72
ISSN - 1541-5856
DOI - 10.4319/lom.2004.2.248
Subject(s) - seawater , dna , chemistry , chromatography , deoxyribonuclease , nucleic acid , trace amounts , biochemistry , biology , ecology , medicine , alternative medicine , pathology
A novel method was developed for the quantification of dissolved deoxyribonucleic acid (D‐DNA) in seawater. This method includes addition of tetrasodium ethylenediamine tetraacetic acid (tetrasodium EDTA) to 0.22 µmfiltered seawater, concentration of > 10 kDa material in the filtrate with a Centricon centrifugal concentration unit, and quantification of the concentrated D‐DNA with the fluorescent double‐stranded DNA stain SYBR Green I. This method requires less than 15 mL of seawater per sample even in oligotrophic environments, and samples can be analyzed in approximately 3 h. The recovery of D‐DNA with this method is 75% to 85% and can be determined for each sample by measuring recovery of 35 S‐labeled DNA added at trace amounts. This method can be used to quantify D‐DNA concentrations as low as 0.01 ng mL −1 with high precision (standard deviation < 5% of the mean). Deoxyribonuclease (DNase) treatment of samples and virus enumeration can be used in conjunction with this method to determine the three major components of D‐DNA: free or enzymatically hydrolyzable DNA (ehD‐DNA), DNA within viruses, and uncharacterized bound DNA.