The Exontrap System - Selective Cloning of Exons. In contrast to bacteria, most eukaryotic genes do not consist of one single continuous amino acids-encoding stretch of DNA, but are split into encoding “exon” parts and large stretches of noncoding “intron” parts. After transcription of the gene into pre- mRNA, the exon sequences are appended to each other by the splicing mechanism resulting in mature mRNA. The classic approach for getting the coding part of a eukaryotic gene is therefore via the isolation of mRNA. Depending on whether there is any sequence information of the gene of interest known, the cDNA can be used for direct cloning by polymerase chain reaction (PCR) or, alternatively, for constructing a cDNA gene bank (e.g., phage gene bank) and further screening. The here described Exontrap System uses an alternative route for the identification of eukaryotic genes. It does not involve an initial isolation of cellular mRNA, but allows the selective cloning of exon sequences from large genomic DNA fragments. One big advantage is that genes, which are not transcribed during certain life cycle stages, can also be identified. Exon/intron mapping is greatly facilitated since for the determination of exon boundaries only the trapped exons have to be sequenced and compared to the known gene. Starting with genomic DNA, an exon library can be derived and screened for cell type-specific gene expression with labeled cDNA from a panel of differentiated cells. The Exontrap System is based on a shuttle vector (E. coli / eukaryotic cells) with an intrinsic splicing function, allowing selective cloning of exon sequences from large genomic eukaryotic DNA fragments. |
