Computational Methods for the Design of PCR Primers for the Amplification of Functional Markers from Environmental Samples

Molecular techniques are becoming increasingly popular for exploring the diversity, function, and structure of microbial communities. Looking at DNA sequences from environmental samples with molecular techniques allows researchers to understand the physiology of organisms that cannot be cultured in the lab. Functional markers are genes specific to a particular metabolic function of interest. For example, ammonia monoxygenase (amo) is a functional marker for nitrification and nitrite reductase (nirS) serves as a marker for denitrification. To assess the diversity of species with a particular metabolic function in a community, functional markers are amplified by PCR, cloned and sequenced (Braker et al., 2000). Functional gene microarrays can then be constructed and used to study community composition (DNA) and functioning (cDNA) (Wu et al., 2001). The PCR amplification of a functional marker requires primers. The design of primers for the amplification of a specific gene from many different species is not a trivial task. The functional markers in the sample can be highly divergent from known sequences, but primers must be very similar to target sequences for efficient amplification. The methods for the design of primers for the amplification of a functional marker from many bacteria in an environmental sample have been ad hoc to date (Braker et al., 1998, Hallin and Lindgren, 1999). This paper reviews the current state of computational methods for PCR primer design and analyzes how these methods with improvements can be incorporated into the design of primers for the amplification of divergent functional markers.