Measurements of DNA base composition with flow cytometry and DNA temperature melting
| Autoři | |
|---|---|
| Rok publikování | 2011 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | Base composition (expressed most frequently as GC content) is an important classification criterion in microbial research. This might be also the case of plants, however, the data on GC contents in plants are still very poor and some skepticism still exist on the reliability of older estimates as well as on the accuracy of recently cumulating data from flow cytometry (FCM) experiments. FCM enables to measure GC content by comparing results with base specific and base unspecific dyes, most frequently unspecific PI and AT-specific DAPI (Barow et Meister, 2002; Bureš et al., 2004; Šmarda et al., 2008). This measurements are critically dependent on the knowledge of binding length of the used base specific dye (i.e., the number of consecutive pairs of the same type enabling binding of a dye molecule), which is, however, mostly uncertain for many dyes and may vary with specific base patterning in a natural DNA. Arbitrary the value of 4 is used for binding length of DAPI in FCM experiments (Barow et Meister, 2002), however, it still remain unclear, whether this approximation is universal across plant phylogeny and to what extent FCM provides reliable estimates of GC content in distantly related taxa with distinct genome organization. Here we tested the reliability of FCM by comparing the results with the parallel measurements done on the same individual with alternative biochemical method of DNA temperature melting (TM) (Marmur et Doty, 1962). This method has been widely used in the past De Ley (1970) and provides GC content estimates similar to that calculated from other biochemical methods and data from the complete genomic sequences. Comparison was done in 14 diverse species of monocots covering wide range of GC contents (33–48%). Parallel measurements with FCM and TM show high degree of correspondence with coefficient of determinacy for ideal regression relationship FCM=TM R2 = 0.935. This correspondence remained very high at widely varying binding lengths and indicates that rather than binding length and variation in base patterning the reliability of the FCM data would be mostly affected with the usage of proper standard with exactly known GC content. |
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