Bauer D, Müller H, Reich J, Riedel H, Ahrenkiel V, Warthoe P, Strauss M
Max-Planck-Gesellschaft, Berlin-Buch, Germany.
Nucleic Acids Res. 1993 Sep 11;21(18):4272-80. doi: 10.1093/nar/21.18.4272.
We have significantly improved a method originally developed by Liang and Pardee [Science 257 (1992) 967-971] to display a broad spectrum of expressed genes and to detect differences in expression between different cell types. We have analysed various aspects of the technique and have modified it for both, the application to fast and efficient identification of genes and the use with automatic analysis systems. Based on the mathematical background we have devised the appropriate number of optimal PCR primers. We have also introduced nondenaturating gels for separating double stranded fragments as single bands. By applying the method to regenerating mouse liver, we have identified, out of a total of 38,000 bands, about 70 fragments where the expression of the corresponding genes seems to be differentially regulated at different time points. Application of the method to an automatic DNA sequencer was successfully done. Thus, we have confirmed the usefulness and increased the power of the RNA display technique, which we named differential display reverse transcription PCR (DDRT-PCR), and have extended the range of its application.
我们对梁和帕迪最初开发的一种方法[《科学》257(1992)967 - 971]进行了显著改进，以展示广泛的表达基因，并检测不同细胞类型之间的表达差异。我们分析了该技术的各个方面，并对其进行了改进，使其既适用于快速高效地鉴定基因，又能与自动分析系统配合使用。基于数学背景，我们设计了合适数量的最佳PCR引物。我们还引入了非变性凝胶来将双链片段分离为单一条带。通过将该方法应用于再生小鼠肝脏，我们在总共38000条带中鉴定出约70个片段，其相应基因的表达在不同时间点似乎受到差异调节。该方法成功应用于自动DNA测序仪。因此，我们证实了RNA展示技术（我们将其命名为差异显示逆转录PCR，DDRT-PCR）的实用性并增强了其功能，同时扩展了其应用范围。
