Senapathy analyzed the distribution of the ORF lengths in computer-generated random DNA sequences first. Surprisingly, this study revealed that about 200 codons (600 bases) was the upper limit in ORF lengths. The shortest ORF (zero base in length) was the most frequent. At increasing lengths of ORFs, their frequency decreased logarithmically, approaching zero at about 600 bases. When the probability of ORF lengths in a random sequence was plotted, it revealed that the probability of increasing lengths of ORFs decreased exponentially and tailed off at a maximum of about 600 bases. From this “negative exponential” distribution of ORF lengths, it was found that most of ORFs were far shorter than the maximum.This finding was surprising because the coding sequence for the average protein length of 400 AAs (with ~1,200 bases of coding sequence) and longer proteins of thousands of AAs (requiring >10,000 bases of coding sequence) would not occur at a stretch in a random sequence. If this was true, a typical gene with a contiguous coding sequence could not originate in a random sequence. Thus, the only possible way that any gene could originate from a random sequence was to split the coding sequence into shorter segments and select these segments from short ORFs available in the random sequence, rather than to increase the ORF length by eliminating consecutive stop codons. This process of choosing short segments of coding sequences from the available ORFs to make a long ORF would lead to a split structure.
If this hypothesis was true, eukaryotic DNA sequences should reflect it. When Senapathy plotted the distribution of ORF lengths in eukaryotic DNA sequences, the plot was remarkably similar to that from random DNA sequences. This plot was also a negative exponential distribution that tailed off at a maximum of about 600 bases, as with eukaryotic genes, which coincided exactly with the maximum length of ORFs observed in both random DNA and eukaryotic DNA sequences.
The split genes thus originated from random DNA sequences by choosing the best of the short coding segments (exons) and splicing them. The intervening intron sequences were left-over vestiges of the random sequences, and thus were earmarked to be removed by the spliceosome. These findings indicated that split genes could have originated from random DNA sequences with exons and introns as they appear in today's eukaryotic organisms. Nobel Laureate Marshall Nirenberg, who deciphered the codons, stated that these findings strongly showed that the split gene theory for the origin of introns and the split structure of genes must be valid.
Blake proposed the Gilbert-Blake hypothesis in 1979 for the origin of introns and stated that Senapathy's split gene theory comprehensively explained the origin of the split gene structure. In addition, he stated that it explained several key questions including the origin of the splicing mechanism: