Relationship between DAPI-fluorescence fading and nuclear DNA content: An alternative method to DNA quantification?

Cristian Gallardo-Escárate,Enrique Dupré,Miguel Ángel Del Río-Portilla,Josué Álvarez-Borrego,Elisabeth Von Brand

doi:10.4067/s0716-97602007000100004

Abstract

In observations by confocal or conventional fluorescence microscopy, important factors should be considered in order to obtain accurate images. One of them, such as the fluorescence bleaching from highest intensity to lowest signal of fluorescence is a common problem with several DNA fluorochromes and especially for DAPI stain. The fluorescence of DAPI fades rapidly when it is exposed to UV light, under optimal conditions of observation. Although the fading process can be retarded using a mounting medium with antifading reagents, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addition, no relationship between fluorescence fading and nuclear DNA content has been tested. In order to test this relationship, we measured by means of image analysis the DAPI-fluorescence intensity in several cellular types (spermatozoa, erythrocytes and haemocytes) during their fluorescence bleaching. An algorithm specifically built in MATLAB software was used for this approach. The correlation coefficient between nuclear DNA content and DAPI-fluorescence fading was found equal to 99%. This study demonstrates the feasibility to measure nuclear DNA content by fluorescence fading quantification, as an alternative method concurrently with image analysis procedures.

Highlights

From the beginning of cytological studies, the amount of nuclear DNA has been considered an indicator of the genetic characteristics of a species, and was taken into account in order to explain the evolutionary process of the species
Image analysis densitometry has been accepted as an accurate means of quantifying DNA for diverse applications (Bertino et al 1994; Gregory 2003), and has been applied in plant and animal genomes (Bennett and Leitch 2001; Gregory 2001)
The use of flow cytometry for measurement of cellular DNA content with high degree of resolution has in recent years been considered as a reliable and standard method

Summary

Introduction

From the beginning of cytological studies, the amount of nuclear DNA has been considered an indicator of the genetic characteristics of a species, and was taken into account in order to explain the evolutionary process of the species. In contrast to the relatively narrow range of genome sizes in prokaryotes, with only approximately a 20fold range among all species measured to date (Kidwell 2002), eukaryotic species vary more than 200,000-fold in the size of their genomes (Gregory 2001) In this scenario, it is generally accepted that the differential amounts of non-coding DNA, such as transposable elements, satellite DNAs, and simple sequence repeats account for a major fraction of eukaryotic genome size variation (Kidwell 2002). Genome sizes have been reported for ~ 3000 animals and nearly 4000 plants, as well as many fungi, protists and bacteria (Bennett and Leitch 2001; Gregory 2001)

Methods

Results

Conclusion