Jarvik J W, Adler S A, Telmer C A, Subramaniam V, Lopez A J
Carnegie Mellon University, Pittsburgh, PA, USA.
Biotechniques. 1996 May;20(5):896-904. doi: 10.2144/96205rr03.
We describe a new method for gene discovery and analysis, CD-tagging, that puts specific molecular tags on a gene, its transcript and its protein product. The method has been successfully tested in two organisms, the haploid unicellular alga Chlamydomonas reinhardtii and the metazoan Drosophila melanogaster. The method utilizes a specially designed DNA molecule, the CD-cassette, that contains splice acceptor and donor sites surrounding a short open reading frame. Insertion of the CD-cassette into an intron in a target gene introduces a new exon, represented by the open reading frame of the CD-cassette, surrounded by two functional hybrid introns. As a result (i) the gene is tagged by a specific nucleotide sequence, (ii) the mRNA is tagged by a specific nucleotide sequence and (iii) the protein is tagged by a specific peptide sequence. Because these tags are unique, specific nucleotide or antibody probes can be used to obtain and/or analyze the gene, transcript or protein. As a gene discovery technology, CD-tagging has two unique advantages: 1) Genes can be identified through a primary screen at the protein level, and so the very process by which a gene is identified provides specific empirical information about its biological function. 2) The cassette arms, which are spliced out of the transcript of the target gene, are available to carry a wide variety of DNA sequences, such as genes encoding drug resistance that can be used to select for the presence of the CD-cassette in the genome.
我们描述了一种用于基因发现和分析的新方法——CD标签法，该方法能在基因、其转录本及其蛋白质产物上添加特定的分子标签。此方法已在两种生物中成功测试，即单倍体单细胞藻类莱茵衣藻和后生动物黑腹果蝇。该方法利用一种特殊设计的DNA分子——CD盒，它包含围绕一个短开放阅读框的剪接受体和供体位点。将CD盒插入目标基因的内含子中会引入一个新的外显子，由CD盒的开放阅读框代表，其两侧是两个功能性杂种内含子。结果是：（i）基因被特定的核苷酸序列标记；（ii）mRNA被特定的核苷酸序列标记；（iii）蛋白质被特定的肽序列标记。由于这些标签是独特的，因此可以使用特定的核苷酸或抗体探针来获取和/或分析基因、转录本或蛋白质。作为一种基因发现技术，CD标签法有两个独特优势：1）可以在蛋白质水平通过初步筛选来鉴定基因，因此鉴定基因的过程本身就能提供有关其生物学功能的特定经验信息。2）从目标基因转录本中剪接出来的盒式臂可用于携带多种DNA序列，例如编码耐药性的基因，可用于筛选基因组中CD盒的存在情况。
