Mapping a mammalian mRNA cap site by restriction digestion of hybridized cDNA

A method using restriction enzymes to locate the cap site in a cloned and sequenced 5' region of a gene is described. With a cDNA fragment (or synthetic oligonucleotide sequence), complementary to any portion of an mRNA, the position of the cap site nucleotide in a genomic clone can be precisely located. The steps include: (1) hybridization of a cDNA fragment to an mRNA sample, and reverse transcription from this primer to produce labeled, fully extended cDNA molecules, (2) hybridization of the extended cDNA to a genomic clone containing the region in which initiation occurs (previously sequenced), (3) restriction endonuclease digestion of the hybrid with two or more enzymes within the putative first exon, and (4) size analysis of the short labeled cDNA fragments produced. Multicut restriction enzymes are most useful in this technique. The sizes of the hybridized fragments correspond to the unique distances from the 5' end of the message to each of the different cleavage sites allowing the cap site to be positioned in the genomic sequence.