Exon amplification: a strategy to isolate mammalian genes based on RNA splicing.
We have developed a method, exon amplification, for fast and efficient isolation of coding sequences from complex mammalian genomic DNA. This method is based on the selection of RNA sequences, exons, which are flanked by functional 5' and 3' splice sites. Fragments of cloned genomic DNA are inserted into an intron, which is flanked by 5' and 3' splice sites of the human immunodeficiency virus 1 tat gene contained within the plasmid pSPL1. COS-7 cells are transfected with these constructs, and the resulting RNA transcripts are processed in vivo. Splice sites of exons contained within the inserted genomic fragment are paired with splice sites of the flanking tat intron. The resulting mature RNA contains the previously unidentified exons, which can then be amplified via RNA-based PCR and cloned. Using this method, we have isolated exon sequences from cloned genomic fragments of the murine Na,K-ATPase alpha 1-subunit gene. We have also screened randomly selected genomic clones known to be derived from a segment of human chromosome 19 and have isolated exon sequences of the DNA repair gene ERCC1. The sensitivity and ease of the exon amplification method permit screening of 20-40 kilobase pairs of genomic DNA in a single transfection. This approach will be extremely useful for rapid identification of mammalian exons and the genes from which they are derived as well as for the generation of chromosomal transcription maps.
Buckler
A J
AJ
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Chang
D D
DD
Graw
S L
SL
Brook
J D
JD
Haber
D A
DA
Sharp
P A
PA
Housman
D E
DE
D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D005091:Exons; D015696:Gene Products, tat / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D012326:RNA Splicing; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D009841:Oligonucleotides; D054322:tat Gene Products, Human Immunodeficiency Virus; D000254:Sodium-Potassium-Exchanging ATPase