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Title: Method of quantitating dsDNA

Abstract

A method for quantitating dsDNA in an aqueous sample solution containing an unknown amount of dsDNA. A first aqueous test solution containing a known amount of a fluorescent dye-dsDNA complex and at least one fluorescence-attenutating contaminant is prepared. The fluorescence intensity of the test solution is measured. The first test solution is diluted by a known amount to provide a second test solution having a known concentration of dsDNA. The fluorescence intensity of the second test solution is measured. Additional diluted test solutions are similarly prepared until a sufficiently dilute test solution having a known amount of dsDNA is prepared that has a fluorescence intensity that is not attenuated upon further dilution. The value of the maximum absorbance of this solution between 200-900 nanometers (nm), referred to herein as the threshold absorbance, is measured. A sample solution having an unknown amount of dsDNA and an absorbance identical to that of the sufficiently dilute test solution at the same chosen wavelength is prepared. Dye is then added to the sample solution to form the fluorescent dye-dsDNA-complex, after which the fluorescence intensity of the sample solution is measured and the quantity of dsDNA in the sample solution is determined. Once the thresholdmore » absorbance of a sample solution obtained from a particular environment has been determined, any similarly prepared sample solution taken from a similar environment and having the same value for the threshold absorbance can be quantified for dsDNA by adding a large excess of dye to the sample solution and measuring its fluorescence intensity.« less

Inventors:
 [1];  [1];  [1]
  1. Los Alamos, NM
Publication Date:
Research Org.:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
OSTI Identifier:
874251
Patent Number(s):
US 6350578
Assignee:
The Regents of the University of California (Los Alamos, NM)
DOE Contract Number:  
W-7405-ENG-36
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
method; quantitating; dsdna; aqueous; sample; solution; containing; amount; fluorescent; dye-dsdna; complex; fluorescence-attenutating; contaminant; prepared; fluorescence; intensity; measured; diluted; provide; concentration; additional; solutions; similarly; sufficiently; dilute; attenuated; dilution; value; maximum; absorbance; 200-900; nanometers; nm; threshold; identical; chosen; wavelength; dye; added; form; dye-dsdna-complex; quantity; determined; obtained; environment; similar; quantified; adding; excess; measuring; fluorescent dye; /435/436/999/

Citation Formats

Stark, Peter C, Kuske, Cheryl R, and Mullen, Kenneth I. Method of quantitating dsDNA. United States: N. p., 2002. Web.
Stark, Peter C, Kuske, Cheryl R, & Mullen, Kenneth I. Method of quantitating dsDNA. United States.
Stark, Peter C, Kuske, Cheryl R, and Mullen, Kenneth I. 2002. "Method of quantitating dsDNA". United States. https://www.osti.gov/servlets/purl/874251.
@article{osti_874251,
title = {Method of quantitating dsDNA},
author = {Stark, Peter C and Kuske, Cheryl R and Mullen, Kenneth I},
abstractNote = {A method for quantitating dsDNA in an aqueous sample solution containing an unknown amount of dsDNA. A first aqueous test solution containing a known amount of a fluorescent dye-dsDNA complex and at least one fluorescence-attenutating contaminant is prepared. The fluorescence intensity of the test solution is measured. The first test solution is diluted by a known amount to provide a second test solution having a known concentration of dsDNA. The fluorescence intensity of the second test solution is measured. Additional diluted test solutions are similarly prepared until a sufficiently dilute test solution having a known amount of dsDNA is prepared that has a fluorescence intensity that is not attenuated upon further dilution. The value of the maximum absorbance of this solution between 200-900 nanometers (nm), referred to herein as the threshold absorbance, is measured. A sample solution having an unknown amount of dsDNA and an absorbance identical to that of the sufficiently dilute test solution at the same chosen wavelength is prepared. Dye is then added to the sample solution to form the fluorescent dye-dsDNA-complex, after which the fluorescence intensity of the sample solution is measured and the quantity of dsDNA in the sample solution is determined. Once the threshold absorbance of a sample solution obtained from a particular environment has been determined, any similarly prepared sample solution taken from a similar environment and having the same value for the threshold absorbance can be quantified for dsDNA by adding a large excess of dye to the sample solution and measuring its fluorescence intensity.},
doi = {},
url = {https://www.osti.gov/biblio/874251}, journal = {},
number = ,
volume = ,
place = {United States},
year = {Tue Jan 01 00:00:00 EST 2002},
month = {Tue Jan 01 00:00:00 EST 2002}
}

Works referenced in this record:

Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification
journal, January 1993


DNA recovery from soils of diverse composition.
journal, January 1996


Characterization of PicoGreen Reagent and Development of a Fluorescence-Based Solution Assay for Double-Stranded DNA Quantitation
journal, July 1997


Recovery of DNA from soils and sediments.
journal, January 1988


Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications
journal, January 1991


Application of the novel nucleic acid dyes YOYO-1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes.
journal, January 1996


Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction.
journal, January 1992


An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis
journal, November 1994


Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.
journal, January 1992


The extraction and purification of microbial DNA from sediments
journal, December 1987


Stable dye–DNA intercalation complexes as reagents for high-sensitivity fluorescence detection
journal, October 1992


Fluorometric Assay Using Dimeric Dyes for Double- and Single-Stranded DNA and RNA with Picogram Sensitivity
journal, January 1993