Abstract：
:DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities. Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quantification of DNA amended with humic substances (20-80 ng/microl) using the Hoechst dye assay was more reliable than with PicoGreen reagent. A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances. In samples containing up to 80 ng/microl of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence. The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample. Vascular plant extract and humic substances reduced the binding of DNA onto the nylon membrane. Low amounts (<2.0 microg) of humic substances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes. DNA samples containing 1.0 microg of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes. Therefore, we suggest that DNA samples containing low concentrations of humic substances (<20 ng/microl) could be used in quantitative membrane hybridization without further purification.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Bachoon DS,Otero E,Hodson REdoi
10.1016/s0167-7012(01)00296-2subject
Has Abstractpub_date
2001-10-01 00:00:00pages
73-82issue
1eissn
0167-7012issn
1872-8359pii
S0167-7012(01)00296-2journal_volume
47pub_type
杂志文章abstract：:A PMA (propidium monoazide) pretreatment protocol, in which PMA is applied directly to membrane filters, was developed for the PCR-based quantification (PMA-qPCR) of viable Legionella pneumophila. Using this method, the amplification of DNA from membrane-damaged L. pneumophila was strongly inhibited for samples contai...
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journal_title：Journal of microbiological methods
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journal_title：Journal of microbiological methods
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pub_type： 杂志文章,评审
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journal_title：Journal of microbiological methods
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journal_title：Journal of microbiological methods
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