• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1561-1569
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• 2006

Denaturing gradient gel electrophoresis (DGGE) is one of the most frequently used methods for analysis of soil microbial community structure. Unbiased PCR amplification of target DNA templates is crucial for efficient detection of multiple microbial populations mixed in soil. In this study, DGGE profiles were compared using different pairs of primers targeting different hypervariable regions of thirteen representative soil bacteria and clones. The primer set (1070f-1392r) for the E. coli numbering 1,071-1,391 region could not resolve all the 16S rDNA fragments of the representative bacteria and clones, and moreover, yielded spurious bands in DGGE profiles. For the E. coli numbering 353-514 region, various forward primers were designed to investigate the efficiency of PCR amplification. A degenerate forward primer (F357IW) often yielded multiple bands for a certain single 16S rDNA fragment in DGGE analysis, whereas nondegenerate primers (338f, F338T2, F338I2) differentially amplified each of the fragments in the mixture according to the position and the number of primer-template mismatches. A forward primer (F352T) designed to have one internal mismatch commonly with all the thirteen 16S rDNA fragments efficiently produced and separated all the target DNA bands with similar intensities in the DGGE profiles. This primer set F352T-519r consistently yielded the best DGGE banding profiles when tested with various soil samples. Touchdown PCR intensified the uneven amplification, and lowering the annealing temperature had no significant effect on the DGGE profiles. These results showed that PCR amplification bias could be much improved by properly designing primers for use in fingerprinting soil bacterial communities with the DGGE technique.

• The Korean Journal of Air & Space Law and Policy
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• v.33 no.2
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• pp.267-290
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• 2018

This paper reviews and criticizes the EU Case of C-452/13, Germanwings GmbH v. Ronny Henning. Under this case, Ronny Henning later sued Lufthansa's budget carrier Germanwings after it refused to pay him 250 euros compensation for a delay he said totalled more than three hours. Germanwings, however, maintained his flight had arrived only two hours and 58 minutes behind schedule. In those circumstances, the following question to the European Court of Justice (ECJ) for a preliminary ruling: What time is relevant for the term time of arrival used in Articles 2, 5 and 7 of Regulation [No 261/2004]: (a) the time that the aircraft lands on the runway (touchdown); (b) the time that the aircraft reaches its parking position and the parking brakes are engaged or the chocks have been applied (in-block time); (c) the time that the aircraft door is opened; (d) a time defined by the parties in the context of party autonomy? ECJ says that the situation of passengers on a flight does not change substantially when their aircraft touches down on the runway at the destination airport, when that aircraft reaches its parking position and the parking brakes are engaged or when the chocks are applied, as the passengers continue to be subject, in the enclosed space in which they are sitting, to various constraints. Therefore, it is only when the passengers are permitted to leave the aircraft and the order is given to that effect to open the doors of the aircraft that the passengers may in principle resume their normal activities without being subject to those constraints. ECJ rules that it is apparent that Articles 2, 5 and 7 of Regulation No 261/2004 must be interpreted as meaning that the concept of 'arrival time', which is used to determine the length of the delay to which passengers on a flight have been subject, corresponds to the time at which at least one of the doors of the aircraft is opened, the assumption being that, at that moment, the passengers are permitted to leave the aircraft.