EXON AMPLIFICATION FROM COMPLETE LIBRARIES OF GENOMIC DNA USING A NOVEL PHAGE VECTOR WITH AUTOMATIC PLASMID EXCISION FACILITY - APPLICATIONTO THE MOUSE NEUROFIBROMATOSIS-1 LOCUS

The identification of transcription units in the vicinity of chromosom al lesions found in tumours is an essential step in the identification of new oncogenes. Here, we describe a lambda phage vector system for genomic exon-trapping (lambda GET), which dramatically simplifies the task of exon amplification from genomic DNA. The vector accommodates a bout 6.5 to 19 kb of DNA and allows inserts to be automatically subclo ned as multi-copy plasmids containing splice donor and acceptor sites positioned flanking the inserted genomic DNA, RNA transcripts derived from such plasmids are processed in vivo and exons contained within th e inserted genomic fragments become flanked by known sequences in the resulting mRNAs. RNA-based PCR can then be used for subsequent cloning and sequence analysis of trapped exons. We have exploited the large c loning capacity of lambda GET to construct highly redundant complete g enomic libraries from Sau3AI partially digested vertebrate DNAs. Using this system, we have analysed a region of about 1 MB around the mouse neurofibromatosis-1 locus and have identified novel transcription uni ts flanking the Nf-1 gene.