Nuclear DNA Content Measurement

Greilhuber, Johann; Temsch, Eva M.; Loureiro, João

doi:10.1002/9783527610921.ch4

Cited by 158 publications

(136 citation statements)

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“…However, this so called external standardization is not recommended, especially in high-precision measurements, as both samples are not treated under identical condition. For the same reason, it is not advisable to mix two independently prepared samples prior to the analysis (45). It has been generally accepted that only the so called internal standardization, when the nuclei of the standard and the unknown sample are isolated, stained, and analyzed simultaneously, yields reliable results (40,(45)(46)(47).…”

Section: Estimation Of Genome Size Using Flow Cytometrymentioning

confidence: 99%

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Nuclear genome size: Are we getting closer?

2010

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Correct information on genome size is important in many areas of research. For a long time, scientists have been struggling to understand the reason for the huge variation in eukaryotic genome size and its biological significance. More recently, the knowledge on genome size has become important to structure genome sequencing projects as their scale and cost depend on genome size. Despite the fact that the first estimates of genome size in eukaryotes were made more than 50 years ago, we are still not quite sure about the exact genome size in practically all animal and plant species. Moreover, different estimates continue to be published for the same species. These discrepancies compromise data comparison and interpretation and point to methodological problems, which include standardization. This article assesses the current state of DNA reference standards for flow cytometry and the issues related to their calibration. ' 2010 International Society for Advancement of CytometryKey terms cytometric techniques; reference standards; genome sequencing; conversion factor; nuclear DNA content; C-value MOST of the genetic information in eukaryotes is localized in the cell nucleus and numerous attempts have been made to determine the quantity of nuclear DNA, especially after various lines of evidence associated DNA with genes (1). It soon became clear that the amount of DNA per nucleus is relatively constant in somatic cells of a given species and that sperm cells have approximately half the DNA amount found for somatic cells (2,3). These experiments provided independent data to support the hereditary role of DNA and marked the beginning of numerous fruitful lines of research and applications, many of them still important today, all relying on the ability to determine DNA amounts in cell nuclei.The early determinations were made colorimetrically on DNA extracted from a known number of cells although the presence of cells in different cell cycle phases could compromise the accuracy of these estimates. The colorimetric approach made identification of subpopulations of cells with different DNA amounts impossible. However, cytometric methods suitable for measurement of absorbance of light in individual nuclei (either using UV light with non-stained nuclei and/or monochromatic visible light with nuclei stained by the Feulgen reaction) were already available during the 1940s and were used to discover the presence of cells with different classes of DNA amounts. Such cells were observed in a variety of animal tissues and were associated with polyploidy and polyteny (4,5). The presence of nuclei with 2, 4, 8, 16, or 32 times the haploid value was described in plant tissues by Swift (6) who introduced the ''C'' terminology to classify nuclear DNA amounts. In this terminology, DNA classes are labeled as C, 2C, 4C, 8C, etc. to characterize DNA amounts of nuclei by multiples of the DNA amount in a complete chromosome set in a non-replicated haploid nucleus, which has the class C DNA amount.At the beginning of the 1950s, biochem...

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Section: Estimation Of Genome Size Using Flow Cytometrymentioning

confidence: 99%

“…Given the human/chicken ratio of 0.357 as determined by Tiersch et al (53), one arrives at a 2C-value of 6.252 pg for human (Ref. 45).…”

Section: Human As a Primary Reference Standardmentioning

confidence: 99%

Nuclear genome size: Are we getting closer?

2010

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“…Flow cytometry is not only faster, but also more precise thanks to the high number of nuclei measured and other characteristics of the technique (Greilhuber et al [16]). There was, furthermore, one genome size of Anemone nemorosa that was unsupported by literature data [17] and that had to be clarified and possibly corrected.…”

Section: Introductionmentioning

confidence: 99%

Heavy Metal Pollution, Selection, and Genome Size: The Species of the Žerjav Study Revisited with Flow Cytometry

Temsch

Ehrendorfer-Schratt

et al. 2010

Journal of Botany

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The Death Valley atŽerjav in northern Slovenia exhibits a gradient of heavy metal pollution in the soil with severe consequences for species richness and composition along this gradient. Recently, a progressive loss of large-genome species in parallel with increasing concentrations of heavy metals has been shown. Here, we have measured the genome size of a near-complete sample of these species with flow cytometry and analysed the correlation of heavy metal pollution with the C-and Cx-values assigned to the test plots. The method of probability analysis was a hypergeometric distribution method. We confirm, on a different methodological basis than previously, that along the pollution gradient, species with high C-and Cx-values are increasingly underrepresented. This lends support to the "large genome constraint hypothesis", predicting that plants with large genomes are at a disadvantage under all aspects of evolution, ecology, and phenotype, because junk DNA imposes a load to the organism.

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“…To protect nucleic acids from nuclease activity, EDTA was used (Dole zel et al, 1989;Marie and Brown, 1993) as a chelating agent for divalent cations, which acted as a nuclease cofactor (Dole zel et al, 1989). Previous reports had shown that PVP decreased the effect of polyphenols by changing their conformational structure, forming hydrogen bonds, and maintaining them in a reduced state (Doyle and Doyle, 1987;Greilhuber et al, 2007;Loureiro et al, 2007b). A higher concentration of the non-ionic detergent Triton X-100 in MB01 buffer facilitated the higher chloroplast lysis, and resulted in a decreased number of fluorescent debris particles (Coba de la Peña and Brown, 2001).…”

Section: Discussionmentioning

confidence: 99%

Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers

Sadhu

Bhadra

Bandyopadhyay

2016

Ann Bot

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Background and Aims Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter-and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues.Methods The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G 0 /G 1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stainnuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01.Key Results Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time.Conclusions Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family.

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Nuclear DNA Content Measurement

Cited by 158 publications

References 93 publications

Nuclear genome size: Are we getting closer?

Nuclear genome size: Are we getting closer?

Heavy Metal Pollution, Selection, and Genome Size: The Species of the Žerjav Study Revisited with Flow Cytometry

Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers

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