Identification and discovery of the coding regions in the genomic structure using computational algorithms is a clear example of such processes.
Except for the 37 kb intron, we mapped the genomic structure using long-range PCR with primers made to the cDNA at regular intervals, and sequenced the amplimers into exon/intron boundaries when introns were detected.
We characterized genomic structure using a combination of existing genomic sequence and direct sequencing from BAC clone template.
Additional file 4 lists all newly annotated OBPs and CSPs along with their CDS and protein sequences, genomic location, and exon structure using BED block size-block start format.
Later, the exact ovine genomic structure was determined using the experimentally derived ovine mRNA sequences.
PCRs were performed on all strains (Table 1) to confirm genomic structure and elements using either Platinum Hi-fidelity PCR Supermix (Invitrogen) or Biotaq (Bioline).
Finally, we analyzed the genomic structure by restriction using the enzyme BssHII to confirm that the synthetic segment was inserted in inverse orientation.
Remarkably, a large amount of polymorphism and structure modifications, indicating intrapopulational polymorphism with not all accessions having an identical genomic structure, was observed using GISH [ 6, 19, 20] and C-banding [ 22- 24] techniques.
Genomic DNA fragments surrounding these miRNA sequences were used to predict the secondary structure using the mfold program [ 38].
A BLAST-based "reciprocal best hit" method, in combination with phylogenetic analysis and genomic structure comparison, was used to determine the orthologous relationships and to identify the COGs.
