Margaret Gentile Computational Methods for the Design of PCR Primers for the Amplification of Functional Markers from Environmental Samples Introduction 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 Lack of computational methods in current designs Studies amplifying sequences from known environmental samples have not been computational to date As a result the results may have underestimated diversity In general known sequences have been globally aligned and primers designed for regions which appear to be conserved The following two studies designed primers for the gene nirS for use in assessing diversity of denitrifiers They illustrate the weakness of current primer design methods In a study by Braker et al primers were designed from conserved sequence segments identified by inspection of six EMBL nirS sequences aligned with MULTIALIGN Braker et al 1998 The specificity of the primers was checked by doing a BLASTN search which revealed significant similarity only to nirS sequences When these primers were used to assess the diversity of denitrifiers in a marine sediment community the resulting clone library contained 228 putative clones few of which were redundant or matched previously seen nirS sequences Braker et al 2000 A similar strategy was employed in a study by Hallin and Lindgren with the addition of adding some degenerate primers to account for some of the wobble positions Hallin and Lindgren 1999 These primers were found to amplify nirS from known denitrifying isolates and did not produce products for non denitrifying isolates Hallin and Lindgren 1999 From these studies it is apparent that primers can be designed which are gene specific and yet are able to amplify a diverse set of sequences for a particular gene What is not clear is whether these primers are able to capture all of the diversity that exists or are merely sampling a subset of the actual diversity present Primer designs relying heavily on consensus nucleotide sequences determined by non computational methods may fail to amplify all of the probably degenerate sequences of a given gene Computational methods for designing PCR primers for a variety of applications have been developed Many of the ideas from these methods could be incorporated into the design of PCR primers for the amplification of degenerate functional markers These methods include calculation of parameters important for primer efficiency such as melting temperature and GC content determination of consensus sequence information more rigorously from local alignments on the protein level and from biological information determination of degenerate nucleotide sequences from probabilistic methods and the use of novel primers composed of consensus and degenerate segments Basics of computational primer design Design Parameters Regardless of the application for which a primer is designed several parameters are used in the design process to quantify its annealing properties and efficiency These parameters include melting temperature GC content and the primer primer interactions The melting temperature is that at which a primer will anneal or break away from the template DNA It depends upon the amino acid sequence and length This temperature is often used as an input for a primer design program because the researcher requires a primer that will work under specified reaction conditions The melting temperature is also important for applications with greater than one primer because primers with different melting temperatures will have different efficiencies One method for calculating melting temperature is the nearest neighbor method Melting temperature is calculated as a function of the sums of the entropy and enthalpy of the consecutive pairs of amino acids Kampke et al 2001 The stability of the primer DNA duplex is important for primer design because it will affect the efficiency of priming The GC content describes the stability of the primer template duplex because different energies are required to break apart GC pairs which have three hydrogen bonds and AT pairs which have only two Kampke et al 2001 Interactions between the forward and reverse primer or a primer with itself are evaluated because these interactions reduce amplification efficiency Algorithms for amplification of known gene The complexity of designing an appropriate primer varies across applications In many applications the DNA sequence is known and the design of primers is simply the identification of an appropriate segment of the known sequence Such applications include sequencing specific gene detection and whole genome microarray construction In sequencing an unknown segment of DNA is amplified for subsequent sequencing by designing primers in known segments that bracket the unknown segment Detecting a gene in a sample is often done by PCR amplification of that gene using primers designed from the known sequence for that gene In whole genome microarray
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