Exon amplification from complete libraries of genomic DNA using a novel phage vector with automatic plasmid excision facility: application to the mouse neurofibromatosis-1 locus.
The identification of transcription units in the vicinity of chromosomal 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 about 6.5 to 19 kb of DNA and allows inserts to be automatically subcloned 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 the 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 cloning capacity of lambda GET to construct highly redundant complete genomic libraries from Sau3AI partially digested vertebrae DNAs. Using this system, we have analysed a region of about 1 MB around the mouse neurofibromatosis-1 locus and have identified novel transcription units flanking the Nf-1 gene.
Department of Medicine 1, University of Freiburg, Germany.
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M
Nehls
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Pfeifer
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T
Boehm
D000595:Amino Acid Sequence; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D005091:Exons; D005784:Gene Amplification; D015723:Gene Library; D016514:Genes, Neurofibromatosis 1; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids