Keohavong P, Gattoni R, Stévenin J
DNA. 1984 Aug;3(4):331-8. doi: 10.1089/dna.1.1984.3.331.
e previously developed a splicing assay (Keohavong et al., 1982) that we designated as a cDNA-RNase assay to analyze the ligation reaction between exons of premessenger RNA during in vivo or in vitro splicing. It was important to determine the specificity of this splicing assay, since the accuracy of in vitro splicing must always be demonstrated clearly. To do this, we constructed DNA probes derived from adenovirus E1A cDNA carrying deletions or insertions of 2-6 bases. After hybridizing them to the wild-type mRNA, the ability of single-strand-specific RNases to detect small mismatches of the RNA-DNA hybrids was examined. The demonstration that an imprecision in the splicing reaction of as little as 2 nucleotides can be detected with an efficiency of 99% indicates the high specificity of the splicing assay and its usefulness for the verification of accurate splicing in in vitro systems.
我们之前开发了一种剪接分析方法（Keohavong等人，1982年），我们将其指定为cDNA-RNase分析方法，用于分析体内或体外剪接过程中前体信使RNA外显子之间的连接反应。确定这种剪接分析方法的特异性很重要，因为体外剪接的准确性必须始终得到明确证明。为此，我们构建了源自腺病毒E1A cDNA的DNA探针，这些探针带有2-6个碱基的缺失或插入。将它们与野生型mRNA杂交后，检测单链特异性核糖核酸酶检测RNA-DNA杂交体小错配的能力。能够以99%的效率检测到剪接反应中低至2个核苷酸的不精确性，这表明剪接分析方法具有很高的特异性，并且对验证体外系统中准确的剪接很有用。
