To ensure specificity of an oligonucleotide probe, its sequence needs to be compared with all other known and predicted gene or transcript sequences of the species and hybridization thermodynamics need to be predicted for all possible cross matches. DNA hybridization thermodynamics depends on sequence composition, relative GC content, number of mismatches, length of consecutive mismatches, and length of gaps. Therefore, 2 cross match alignments having similar length and number of identities may differ greatly in their cross hybridization thermodynamics.

This can be easily seen from following example:

Thus, conventional sequence similarity algorithms e.g. BLAST may have limitations in predicting cross-hybridization possibilities. Therefore, a proprietary sequence similarity search method has been employed in Sarani for fast computation of all thermodynamically relevant cross matches. The motivations behind development and implementation of this algoritm are:

• To compute alignments with the objective of estimating possible cross hybridization effects rather than sequence similarity alone.
• To use scoring schemes, maximal consecutive mismatches and gap penalty in tune with DNA hybridization thermodynamics based on nearest neighbour energy parameters.
• Complete integration of sequence similarity search with the oligo design process.

  Strand Local Alignment Search Tool (SLAST)

Strand Local Alignment Search Tool is a BLAST (blastn) like tool, tailored with appropriate data-structures to suit the measurement of specificity of an oligo while doing large scale oligonucleotide probe design. Sarani's sequence similarity search algorithm differs from BLAST basically in two ways:

• To obtain an HSP, BLAST uses an automaton based approach, while SLAST uses a hash table based search.
• The second difference is the extension of an HSP. The stopping criterion in BLAST for extending an HSP is based on the sequence alignment score. In SLAST, the stopping criterion is influenced by the thermodynamic stability of the alignment. SLAST stops extending an HSP when the melting temperature of the alignment no longer becomes cumulative in terms of the base-pairs.