Under the split gene theory, an exon is defined by an ORF. It requires a mechanism to recognize an ORF to have originated. As an ORF is defined by a contiguous coding sequence bounded by stop codons, these stop codon ends had to be recognized by the exon-intron gene recognition system. This system could have defined the exons by the presence of a stop codon at the ends of ORFs, which should be included within the ends of the introns and eliminated by the splicing process. Thus, the introns should contain a stop codon at their ends, which would be part of the splice junction sequences.
If this hypothesis was true, the split genes of today's living organisms should contain stop codons exactly at the ends of introns. When Senapathy tested this hypothesis in the splice junctions of eukaryotic genes, he found that the vast majority of splice junctions did contain a stop codon at the end of each intron, outside of the exons. In fact, these stop codons were found to form the “canonical” GT:AG splicing sequence, with the three stop codons occurring as part of the strong consensus signals. Thus, the basic split gene theory for the origin of introns and the split gene structure led to the understanding that the splice junctions originated from the stop codons.
Sequence data for only about 1,000 exon-intron junctions were available when Senapathy thought about this question. He took the data for 1,030 splice junction sequences (donors and acceptors) and counted the codons occurring at 
each of the 7- base positions in the donor signal sequence [CAG:GTGAGT] and each of the possible 2-base positions in the acceptor signal [CAG:G] from the GenBank database. He found that the stop codons occurred at high frequency only at the 5th base position in the donor signal and the first base position in the acceptor signal. These positions are the* start of the intron (in fact, one base after the start) and at the end of the intron, as Senapathy had predicted. The codon counts at only these positions are shown. Even when the codons at these positions were not stop 
codons, 70% of them began with the first two bases of the stop codons TA and TG [TAT = 75; TAC = 59; TGT = 70].
All three stop codons (TGA, TAA and TAG) were found after one base (G) at the start of introns. These stop codons are shown in the consensus canonical donor splice junction as AG:GT(A/G)GGT, wherein the TAA and TGA are the stop codons, and the additional TAG is also present at this position. Besides the codon CAG, only TAG, which is a stop codon, was found at the ends of introns. The canonical acceptor splice junction is shown as (C/T)AG:GT, in which TAG is the stop codon. These consensus sequences clearly show the presence of the stop codons at the ends of introns bordering the exons in all eukaryotic genes, thus providing a strong corroboration for the split gene theory. Nirenberg again stated that these observations fully supported the split gene theory for the origin of splice junction sequences from stop codons.
Soon after the discovery of introns by Philip Sharp and Richard Roberts, it became known that mutations within splice junctions could lead to diseases. Senapathy showed that mutations in the stop codon bases (canonical bases) caused more diseases than the mutations in non-canonical bases.