Degenerate Primer Design for Highly Variable Genomes
- 5.3k Downloads
The application of degenerate PCR primers towards target amplification and sequencing is a useful technique when a population of organisms under investigation is evolving rapidly, or is highly diverse. Degenerate bases in these primers are specified with ambiguity codes that represent alternative nucleotide configurations. Degenerate PCR primers allow the simultaneous amplification of a heterogeneous population by providing a mixture of PCR primers each of which anneal to an alternative genotype found in the isolated sample. However, as the number of degenerate bases specified in a pair of primers rises, the likelihood of amplifying unwanted alternative products also increases. These alternative products may confound downstream data analyses if their levels begin to obfuscate the desired PCR products. This chapter describes a set of computational methodologies that may be used to minimize the degeneracy of designed primers, while still maximizing the proportion of genotypes assayed in the targeted population.
Key wordsDegenerate PCR primers Primer design Mixed population PCR Targeted resequencing Viral genome amplification Hierarchical clustering Population stratification
The development of the degenerate primer design consensus sequence construction strategy was funded in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and Department of Health and Human Services under contract number HHSN272200900007C. This work was supported by the Office of Biological and Environmental Research in the DOE Office of Science award DESC0006837.