Margaret GentileComputational Methods for the Design of PCR Primersfor the Amplification of Functional Markers fromEnvironmental SamplesIntroductionMolecular techniques are becoming increasingly popular for exploring thediversity, function, and structure of microbial communities. Looking at DNA sequencesfrom environmental samples with molecular techniques allows researchers to understandthe physiology of organisms that cannot be cultured in the lab. Functional markers aregenes specific to a particular metabolic function of interest. For example, ammoniamonoxygenase (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 particularmetabolic function in a community, functional markers are amplified by PCR, cloned andsequenced (Braker et al., 2000). Functional gene microarrays can then be constructedand used to study community composition (DNA) and functioning (cDNA) (Wu et al.,2001).The PCR amplification of a functional marker requires primers. The design ofprimers for the amplification of a specific gene from many different species is not atrivial task. The functional markers in the sample can be highly divergent from knownsequences, but primers must be very similar to target sequences for efficientamplification. The methods for the design of primers for the amplification of a functionalmarker from many bacteria in an environmental sample have been ad hoc to date (Brakeret al., 1998, Hallin and Lindgren, 1999). This paper reviews the current state ofcomputational methods for PCR primer design and analyzes how these methods withimprovements can be incorporated into the design of primers for the amplification ofdivergent functional markers.Lack of computational methods in current designsStudies amplifying sequences from known environmental samples have not beencomputational to date. As a result, the results may have underestimated diversity. Ingeneral, known sequences have been globally aligned , and primers designed for regionswhich appear to be conserved. The following two studies designed primers for the genenirS for use in assessing diversity of denitrifiers. They illustrate the weakness of currentprimer design methods.In a study by Braker et al., primers were designed from conserved sequencesegments identified by inspection of six EMBL nirS sequences aligned withMULTIALIGN. (Braker et al., 1998). The specificity of the primers was checked bydoing 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 sedimentcommunity, the resulting clone library contained 228 putative clones, few of which wereredundant or matched previously seen nirS sequences. (Braker et al, 2000) A similarstrategy was employed in a study by Hallin and Lindgren with the addition of addingsome degenerate primers to account for some of the wobble positions. (Hallin andLindgren, 1999) These primers were found to amplify nirS from known denitrifyingisolates 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 genespecific and yet are able to amplify a diverse set of sequences for a particular gene. Whatis not clear is whether these primers are able to capture all of the diversity that exists orare merely sampling a subset of the actual diversity present. Primer designs relyingheavily on consensus nucleotide sequences determined by non-computational methodsmay fail to amplify all of the probably degenerate sequences of a given gene.Computational methods for designing PCR primers for a variety of applications havebeen developed. Many of the ideas from these methods could be incorporated into thedesign of PCR primers for the amplification of degenerate functional markers. Thesemethods include; calculation of parameters important for primer efficiency such asmelting temperature and GC content, determination of consensus sequence informationmore rigorously from local alignments on the protein level and from biologicalinformation, determination of degenerate nucleotide sequences from probabilisticmethods, and the use of novel primers composed of consensus and degenerate segments.Basics of computational primer designDesign ParametersRegardless of the application for which a primer is designed, several parametersare used in the design process to quantify its annealing properties and efficiency. Theseparameters 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 thetemplate DNA. It depends upon the amino acid sequence and length. This temperatureis often used as an input for a primer design program, because the researcher requires aprimer that will work under specified reaction conditions. The melting temperature isalso important for applications with greater than one primer, because primers withdifferent melting temperatures will have different efficiencies. One method forcalculating melting temperature is the nearest neighbor method. Melting temperature iscalculated as a function of the sums of the entropy and enthalpy of the consecutive pairsof amino acids (Kampke et al., 2001). The stability of the primer DNA duplex isimportant for primer design, because it will affect the efficiency of priming. The GCcontent describes the stability of the primer template duplex, because different energiesare required to break apart GC pairs which have three hydrogen bonds and AT pairswhich have only two (Kampke et al., 2001). Interactions between the forward andreverse primer or a primer with itself are evaluated, because these interactions reduceamplification efficiency.Algorithms for amplification of known geneThe complexity of designing an appropriate primer varies across applications. Inmany applications, the DNA sequence is known, and the design of primers is simply theidentification of an appropriate segment of the known sequence. Such applicationsinclude sequencing, specific gene detection, and whole genome microarray construction.In sequencing, an unknown segment of DNA is amplified for subsequent sequencing bydesigning primers in known segments that bracket the unknown segment. Detecting agene in a sample is often done by PCR amplification of that gene using primers
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