If you are working with a eukaryote, you may wish to see if two or more neighboring exons are part of the same gene. If this is the case, you should be able to locate a donor splice at the 3’ end of one exon and an acceptor splice at the 5’ end of the next. You can search for acceptor and donor splices using the Patterns – Matrix method. Matrix pattern files are available in both “donor” and “acceptor” varieties for a number of organisms. This is the same method used for predicting start sites, except that distinct matrix pattern files are used for start sites vs. donor splice sites vs. acceptor splice sites.
To jump to sites containing the highly conserved core residues of the splice pattern:
First select the range of the sequence you want to work in. Then choose Edit > Move Selection, Edit > Find/Move Next and Edit > Find/Move Previous to jump through the sites. Once you have located splice sites, you may annotate the feature and join related exons together to form a putative gene.
To confirm the annotated feature has no internal stop codons:
Select the feature and choose Analysis > Codon Usage. A table summarizing the frequency of occurrence of all codons in the selected feature appears.
To seek evidence for the terminus of a coding region:
Do any of the following:
- Seek stop codons.
- Correlate the falloff in the last of a series of Borodovsky peaks with the stop codon for the corresponding ORF. In eukaryotes, you may also seek an ORF and corresponding Borodovsky peak for which there are no compelling splice sites available for a subsequent exon.
- Use the Patterns – Type-in Pattern method to seek nearby potential polyadenylation signals on the appropriate strand.
The next step is to attempt to ascertain the identity of your gene via a BLAST search.
To align exons to a reference sequence for corroboration:
This procedure is demonstrated in the following video:
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