Related Experiment Video
Updated: Jul 13, 2026
07:44
Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye
Published on: November 5, 2010
Quantifying DNA concentrations using fluorometry: a comparison of fluorophores
Kalpana Rengarajan1, Stphen M Cristol, Milan Mehta
1Department of Ophthalmology, Emory Eye Center, Emory University, Atlanta, GA 30322, USA.
Molecular Vision
|November 15, 2002
Summary
Newer fluorophores like SYBR Green I and PicoGreen offer superior sensitivity for quantifying double-stranded DNA (dsDNA) in scant ocular tissue samples, crucial for vision research.
Area of Science:
- Biochemistry
- Molecular Biology
- Ophthalmology
Background:
- Accurate DNA quantification is vital in vision research, particularly with limited ocular tissue samples from animal models.
- Fluorometry offers a sensitive and rapid method for analyzing DNA concentration, surpassing traditional absorption spectrometry.
Purpose of the Study:
- To compare the sensitivity of various fluorophores for quantifying double-stranded DNA (dsDNA) concentrations using fluorometry.
- To identify the most effective fluorophores for detecting low DNA concentrations in biological samples.
Main Methods:
- Quantitative analysis of dsDNA concentrations using fluorometry.
- Comparative assessment of fluorophore sensitivity: SYBR Green I, PicoGreen, ethidium bromide, and Hoechst 33258.
Main Results:
- SYBR Green I and PicoGreen demonstrated significantly higher sensitivity in quantifying DNA concentrations compared to ethidium bromide and Hoechst 33258.
- Fluorometry with optimized fluorophores enables reliable detection of low dsDNA levels.
Conclusions:
- SYBR Green I and PicoGreen are recommended as highly sensitive fluorophores for dsDNA quantification in vision research.
- Fluorometric methods provide a sensitive and efficient approach for analyzing limited DNA samples in biological research.
Related Concept Videos
03:31Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
02:34DNA Microarrays
Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...
02:57Real Time RT-PCR
Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
The real-time quantification of the number of amplified products is...
01:25Fluorescence and Phosphorescence: Instrumentation
Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.