Degenerate Primers
Degeneracy in primer sequence should also be taken into consideration. In fact researchers pursuing the cloning of novel genes often face the problem that only a partial protein sequence is known (Bindon et al., 1998). In these circumstances several procedures can be used, some involve universal primers or reverse translation of the protein sequence into a DNA sequence and the design of primers from this sequence. However, due to redundancy in the genetic code, primer design must account for the ambiguous DNA bases and has to be designed in the region of lowest degeneracy (Kwok et al., 1994). Le Guyader et al. (1996) evaluated the effect of degenerate primers in the detection of caliciviruses. Mack and Sninsky (1988) demonstrated the selection of conserved regions encoded by amino acids with minimal codon degeneracy in order to reduce mismatch. Degenerate primers based on the amino acid sequence of conserved regions were also used to search for members of a gene family (Wilks et al., 1989), homologous genes from different species (Kopin et al., 1990) and related viruses (Mack and Sninsky, 1988; Manos et al., 1989; Shih et al., 1989). A computer program was also developed specifically for degenerate primer design (Chen and Zhu, 1997).
Software in Primer Design
Most molecular biological applications are aided by software. The use of software in biological applications has given a new dimension to the field of bioinformatics. Many different programs for the design of primers are now available. Freeware software is available on the internet and many universities have established servers where a user can log on and perform free analyses of proteins and nucleic acid sequences. There are number of simple stand-alone programs as well as complex integrated networked versions of the commercial software available. These software packages may be for complete DNA and protein analysis, secondary structure predictions, primer design, molecular modeling, development of cloning strategies, plasmid drawing or restriction enzyme analyses etc. Companies engaged in biosoftware development include: Alkami Biosystems, Molecular Biology Insights, PREMIER Biosoft International, IntelliGenetics Inc., Hitachi Inc., DNA Star, Advanced American Biotechnology and Imaging.
Some scientists have also developed algorithms and computer programs for various purposes of primer design (Rychlik and Rhoades, 1989; Lowe et al., 1990; Lucas et al., 1991; O'Hara and Venezia, 1991; Tamura et al., 1991; Makarova et al., 1992; Osborne, 1992; Plasterer, 1997; Sze et al., 1998).
Conclusion
Biological science, and in particular biotechnology, is rapidly changing and cannot achieve its objectives without the help of computer technology and information technology tools. PCR primer design concepts are not new. However constant upgrading and updating of the strategies and methods are essential to maintain rapid and efficient progress. Computational strategies in biotechnology are of particular importance. The algorithms relevant to the efficient design of primers should be modified taking into account experimental data.
References
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