Yoshikawa Y, Mukai H, Asada K, Hino F, Kato I
Advanced Biomedical Center, Takara Shuzo Co., Ltd., Shiga, Japan.
Anal Biochem. 1998 Feb 1;256(1):82-91. doi: 10.1006/abio.1997.2473.
Differential display (DD) has been used extensively to detect differentially expressed genes. However, the low reproducibility of displayed bands makes verification by Northern blot difficult and the technique is labor-intensive. This report describes a fluorescent DD with a ROX (carboxy-X-rhodamine)-labeled anchor primer and a revised RT-PCR, utilizing AMV reverse transcriptase, a more thermostable reverse transcriptase than Mu-MLV, and optimized concentrations of dNTPs and of MgCl2. Our technique yielded clear fingerprints with high reproducibility. Further, we have developed a method of rapid screening to select the cDNA fragments of interest in excised bands from a polyacrylamide gel without cloning. This method consists of electrophoresis with an agarose gel containing a base-specific DNA ligand to separate the equally sized fragments differing in base composition, and side-by-side comparison of the reamplified products from the experimental and control lane. Most of the cDNA fragments selected by this protocol provided readable sequences by direct sequencing and were confirmed to exhibit differential expression by Northern blot analysis or semiquantitative RT-PCR.
差异显示（DD）已被广泛用于检测差异表达基因。然而，所显示条带的低重现性使得通过Northern印迹进行验证变得困难，并且该技术劳动强度大。本报告描述了一种荧光差异显示方法，使用ROX（羧基-X-罗丹明）标记的锚定引物和改进的RT-PCR，利用AMV逆转录酶（一种比Mu-MLV更耐热的逆转录酶）以及优化浓度的dNTP和MgCl2。我们的技术产生了具有高重现性的清晰指纹图谱。此外，我们开发了一种快速筛选方法，无需克隆即可从聚丙烯酰胺凝胶中切下的条带中选择感兴趣的cDNA片段。该方法包括在含有碱基特异性DNA配体的琼脂糖凝胶上进行电泳，以分离碱基组成不同但大小相同的片段，并对实验泳道和对照泳道重新扩增的产物进行并排比较。通过该方案选择的大多数cDNA片段通过直接测序提供了可读序列，并通过Northern印迹分析或半定量RT-PCR证实表现出差异表达。
