Rinaldy A R, Steiner M S
University of Tennessee Urologic Research Laboratories, College of Medicine, University of Tennessee, Memphis 38163, USA.
DNA Cell Biol. 1999 Nov;18(11):829-36. doi: 10.1089/104454999314827.
A technique to improve cDNA library screening was developed by using mixed probes derived from two closely related cDNA populations of high-metastatic MAT-LyLu and low-metastatic AT-1 Dunning R3227 rat prostate cancer sublines. The technique required the generation of a cDNA library from each subline followed by polymerase chain reaction (PCR) amplification of the cDNA insert population. The PCR products derived from the first library were radiolabeled and mixed with an excess amount of PCR products from the second library. The mixture and an excess amount of both the lambda and pBluescript DNA were used as a probe to screen the first cDNA library. This mixed probe (designated the competition probe) differentially cross-hybridized with the plaque lift of the screened first cDNA library. Weak radioactive signals indicated the cross-hybridization of cDNA sequences common to the competition probe mixture and the first cDNA library, whereas strong signals implied unhybridized unique or abundant cDNA sequences in the first cDNA library. The reproducibility of this technique was confirmed by showing that the full-length cDNA clones were associated with the phenotype of the screened first cell line. The isolated clones were characterized as rat nucleolar protein, rat mitochondrial genes coding for 16S and 12S rRNAs, and rat tRNAs specific for valine and phenyl-alanine. This result is consistent with the fact that the first cell line, MAT-LyLu, is metabolically more active than are AT-1 cells because of higher gene dosage or amplification of nucleolar and mitochondrial RNA and its associated genes. Another clone which had a strong signal represented a novel gene associated with the MAT-LyLu cancer phenotype.
通过使用源自高转移性MAT-LyLu和低转移性AT-1 Dunning R3227大鼠前列腺癌亚系两个密切相关的cDNA群体的混合探针，开发了一种改进cDNA文库筛选的技术。该技术需要从每个亚系生成一个cDNA文库，然后对cDNA插入片段群体进行聚合酶链反应（PCR）扩增。将源自第一个文库的PCR产物进行放射性标记，并与过量的来自第二个文库的PCR产物混合。该混合物以及过量的λ噬菌体和pBluescript DNA用作探针来筛选第一个cDNA文库。这种混合探针（称为竞争探针）与筛选的第一个cDNA文库的菌斑杂交信号存在差异。弱放射性信号表明竞争探针混合物与第一个cDNA文库共有的cDNA序列发生了交叉杂交，而强信号则意味着第一个cDNA文库中未杂交的独特或丰富的cDNA序列。通过显示全长cDNA克隆与筛选的第一个细胞系的表型相关，证实了该技术的可重复性。分离的克隆被鉴定为大鼠核仁蛋白、编码16S和12S rRNA的大鼠线粒体基因以及缬氨酸和苯丙氨酸特异性的大鼠tRNA。这一结果与以下事实一致，即第一个细胞系MAT-LyLu由于核仁及线粒体RNA及其相关基因的基因剂量更高或扩增，其代谢活性比AT-1细胞更高。另一个具有强信号的克隆代表了一个与MAT-LyLu癌症表型相关的新基因。
