The homology between two exons in a pair was identified using the bl2seq implementation of TBLASTN.
If the distance between two exons was less than 10 bases, the two exons would be merged together.
Primers were placed at the junction between two exons.
To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between two exons or on two different exons.
When possible, primers or probes were designed to span between two exons.
In genes containing between two and ten exons, the SC frequencies showed arched-curves ('∩' or '∪'), interstitial exons had higher or lower frequencies than two terminal exons from the first to the last exons in both algae and land plants (data not shown).
Using the RUM_Unique and splice junction files, 14,696 novel internal exons (novel exons found between two annotated exons), exon skipping, and alternate 3′/5′ splice sites were selected for high-throughput validation.
For example, if exon 2 of gene A starts at position 13,780 and ends at 13,942 on Chromosome 1, and exon 5 of gene B starts at 13,950 and ends at 13,820 on the same chromosome, we can infer that these two genes form a pair of sas overlapping genes and that the overlapping region between the two exons has (13942−13820+1) = 123 bp.
To study the coordination between alternative exons, we performed a large scale analysis of the correlation (coordination) between the two exons in an adjacent cassette exon pair.
The exon junctions are perfectly conserved for PPARG exon 5 and TSEN2 exon 2 and the 174 bp region between the two exons on the EST sequence appears to correspond on the human genome to a LINE L2 interspersed repeat.
