Pre-PCR DNA quantitation of soil and sediment samples: Method development and instrument design

P. C. Stark, K. I. Mullen, K. Banton, R. Russotti, D. Soran, C. R. Kuske

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

A simple and straightforward method for the quantitation of dsDNA in soil and sediment matrices has been developed to support rapid, in-the-field PCR analysis of environmental samples. This method uses PicoGreen nucleic acid stain, and a combination of UV/Vis and fluorescence spectroscopy, to quantitate dsDNA in the presence of interfering humic materials. The practical utility of this approach is that it compliments a seven-step DNA extraction procedure for environmental samples. The DNA quantitation method is utilized twice during the extraction procedure. Once, prior to a micro-spin column procedure to maximize the amount of DNA extracted, and a second time, just prior to PCR to optimize the PCR reaction conditions. A field-portable, assay-specific instrument has been developed based on this methodology. Software for this instrument steps the analyst through the experimental procedure, and has been designed such that a minimum of technical expertise is required to perform the assay. Initial data obtained from the prototype unit indicates that this instrument compares with commercial instrumentation in terms of detection limit and sensitivity. (C) 2000 Elsevier Science Ltd.

Original languageEnglish (US)
Pages (from-to)1101-1110
Number of pages10
JournalSoil Biology and Biochemistry
Volume32
Issue number8-9
DOIs
StatePublished - Aug 1 2000

Bibliographical note

Funding Information:
This work was sponsored by the US Government, in particular a research project sponsored by J. Dorigan.

Keywords

  • DNA quantitation
  • Field-portable
  • Fluorescence
  • Humic materials
  • PicoGreen
  • Polymerase chain reaction
  • UV/Vis absorption

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