br Conclusion As far as we know this work
Conclusion As far as we know, this work constituted the first use of silver nitrate staining and sequential FISH in V. vinifera. This technique allowed the unequivocal identification of Ag-NORs and corresponding rDNA loci. Besides, silver nitrate staining and sequential FISH may bring new insights about the variation in number of transcriptionally active rDNA loci and differential nucleolar activity among V. vinifera varieties.
Conflicts of interest
Acknowledgements The authors CC and AC thank their grants BI/INTERACT/VW/183/2016 and BPD/UTAD/INNOVINE&WINE/593/2016, respectively, both supported by the NORTE-01-0145-FEDER. The author IP thanks his doctoral grant PD/BD/113611/2015 attributed by the Portuguese Foundation for Science and Technology (FCT) under the scope of the Doctoral Program “Agricultural Production Chains – from fork to farm” (PD/00122/2012).
Introduction The ability to extract and PCR amplify DNA from biological stains is a key (±)-J 113397 in Forensic Genetics. Fortunately DNA often survives for months or years at ambient conditions in untreated dry stains.1, 2, 3, 4, 5, 6 The effects of various parameters on the survival of DNA, such as UV-light, stain substrates, admixture of soil, age, humidity, and temperature have been previously assessed.2, 6, 7, 8, 9 However, to the knowledge of the authors there is little information about the stability of DNA in stains when controlled conditions of humidity and temperature approach the extreme. Microbial enzymatic attack is of fundamental importance in the degradation of organic matter but depends, among else, on the availability of water. Also many of the non-enzymatic processes that degrade DNA depend on the presence of water.11, 12A priori one would therefore expect an accelerated rate of degradation of DNA in biological stains as the relative humidity approaches 100%. To determine the range of natural conditions that allow an adequate stability of DNA in stain material, blood stains without any stabilizing additives were incubated at various conditions of relative humidity (RH) and temperature. The quality and quantity of the remaining DNA was estimated by the ability to PCR amplify long and short fragments and by quantitative assessment using real time PCR.
Materials and methods
Results In all four independent series (I–IV) DNA incubated at room temperature remained amplifiable for the entire experimental period as long as RH was less than 100% (93%, 80% and 50%). The same was the case at 35 °C for three of the four series (II–IV) while DNA in series I remained stable up to seven month (Fig. 1, Fig. 2). At 100% RH the survival time was shorter, down to approximately four months for the 273bp fragment, and approximately two months for the 1600bp fragment, but large differences were observed among the series, and in one series (II) the short fragment remained amplifiable for the entire experimental period (Fig. 2). Microbial growth was observed at 100% RH after various amounts of time (1½–9 months), but in most cases growth only occurred on some of the filter paper pieces (Fig. 2 and Table S2). The occurrence of microbial growth was not more pronounced in series III, despite the fact that this series had been deliberately exposed to out-door conditions. Stains with microbial growth showed little or no surviving human DNA. Most important, growth was not observed on any of the stains incubated at 93% RH or less. The stability of DNA at 100% RH was further tested at higher temperatures, 45–65 °C (series V, Fig. 3). At 45 °C the amplification of both fragments was still possible after eight to nine months. However, at 55 °C and 65 °C the stability dropped considerable, the 273bp fragment was only detectable for three months, and amplification of the 1600bp fragment was not possible after one month. Microbial growth was not observed at any of these elevated temperatures. Real time PCR assessment of the quantity of intact DNA at selected time points was performed for series II–IV (Table S2). Although the number of extracted templates showed a considerable variation among the stains, the results generally paralleled the strength of the corresponding electrophoretic bands. The extracts of stains taken at time 0 showed the least variation among the series (range 329–443 copies/5 μl extract) and served as extraction controls. Since the optimal number of templates that was obtainable in five μl extract was ∼2000 copies (see Materials and methods), the extraction efficiency with fresh stains was only about 20%. Extraction of DNA using Qiagen silica-membrane-based spin columns may result in lower yields of DNA compared to other methods,16, 17 however, the QIAamp® DNA Mini Kit was chosen for the present work since it is a standardized and widely used method. It is noted that some extracts from later time points showed higher values. Whether this observation is caused by an increase in the extraction efficiency of older stains (e.g., because of decomposition of proteins and lipids trapping the nuclear DNA) or simply reflects a high variance between stains remains unresolved. However, overall the quantitative results confirmed that the stains contained sufficient DNA for adequate PCR amplification after 12 month at 50–93% RH at room temperature or 35 °C (>16,000 or >8200 copies of DNA/5 μl blood, respectively).