Mapping introns by exon excision

Abstract

A direct method for mapping introns has been devised. The technique makes use of a radioactive synthetic RNA transcript of the gene and a complementary, single-stranded DNA copy of mRNA-derived sequences. Upon hybridization of the cDNA to RNA and cleavage with ribonuclease H, only exonic RNA sequences are degraded. The surviving RNA fragments are the introns. Electrophoretic analysis in denaturing agarose gels reveals the number and size of the introns. The order of the introns is determined separately using unlabeled RNA transcripts; surviving RNA fragments are transferred to a solid support and the blot is probed sequentially with a nested set of genomic RNA transcripts of the opposite strand. Using the human prothymosin α gene as an example, four introns were identified which from 5′ to 3′ were 2.6, 0.47, 0.47, and 0.28 kb in size. From mapping and sequencing experiments the sizes are 2.6, 0.465, 0.459, and 0.295 kb, respectively. Similarly, the presence of two 300-bp insertions in a human prothymosin α pseudogene was established; the inserts were later identified as 295-bp Alu repetitive elements.