Köhrer K, Kutchan T M, Domdey H
Laboratorium für Molekulare Biologie-Genzentrum-der Ludwig-Maximilians-Universität München, Martinsried, FRG.
DNA. 1989 Mar;8(2):143-7. doi: 10.1089/dna.1.1989.8.143.
By combining several established techniques we developed a method to test the specificity of mixed oligodeoxynucleotide hybridization probes and to provide the information for the design of long nondegenerate, and therefore more specific probes. Mixed oligodeoxynucleotide probes derived from known peptide sequences are first used to initiate primer extension reactions with poly(A)+RNA as template in the presence of three dNTPs and one ddNTP to generate cDNA transcripts of defined lengths. Comparing the lengths of the cDNA transcripts with the possible nucleic acid sequence coding for the known oligopeptide indicates whether the oligodeoxynucleotide mix hybridizes predominantly to the RNA of interest. In a second step, the oligodeoxynucleotide mix with the highest specificity is used for indirect RNA sequence analysis. This confirms the specificity of the probe and provides information to design a long, highly specific oligodeoxynucleotide probe for the gene of interest. This simple two-step-procedure helps to circumvent the time-consuming procedures of subcloning and sequencing of cross-hybridizing fragments.
通过结合几种成熟的技术，我们开发了一种方法来测试混合寡脱氧核苷酸杂交探针的特异性，并为设计长的、非简并的、因此更特异的探针提供信息。首先，将源自已知肽序列的混合寡脱氧核苷酸探针与聚腺苷酸加尾RNA（poly(A)+RNA）作为模板，在三种脱氧核苷酸三磷酸（dNTP）和一种双脱氧核苷酸三磷酸（ddNTP）存在的情况下启动引物延伸反应，以生成特定长度的互补DNA（cDNA）转录本。将cDNA转录本的长度与编码已知寡肽的可能核酸序列进行比较，可表明寡脱氧核苷酸混合物是否主要与目标RNA杂交。在第二步中，将具有最高特异性的寡脱氧核苷酸混合物用于间接RNA序列分析。这证实了探针的特异性，并为设计针对目标基因的长的、高度特异的寡脱氧核苷酸探针提供信息。这种简单的两步法有助于规避对交叉杂交片段进行亚克隆和测序的耗时过程。
