SPADA Docs

225 Figure 5. Traditional primer design approach. MSA: Multiple sequence alignment. The software 226 tools depicted are only exemplar suggestions and not an endorsement of specific tools. Any other 227 software with an equivalent functionality can also be used for producing similar outputs. 228 4.3 Modern Primer Design Paradigm 229 4.3.1 Modern sequence databases 230 Perhaps the most important new contribution to the field of PCR is the availability of modern 231 sequence databases – they are a treasure trove that can be used to improve the design of the PCR 232 assay to maximize coverage (i.e. the number of variants that are efficiently amplified by a given 233 PCR reaction) and also guide the testing of a PCR assay by identifying potential false positives 234 or false negatives (e.g., due to sequence variations at primer and probe sites). When designing a 235 PCR assay, it is helpful to first collect sets of sequences that represent the inclusivity, 236 exclusivity, and background panels ( see Glossary for definitions). In addition to the generalized 237 databases such as National Center for Biotechnology Information (NCBI) GenBank, European 238 Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) and the DNA 239 Data Bank of Japan (DDBJ), there are a variety of invaluable curated pathogen genome 240

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