Degenerate Primers

DegeneratePrimers

Foramplificationofcognatesequencesfromdifferentorganisms,orfor"evolutionaryPCR",onemayincreasethechancesofgettingproductbydesigning"degenerate"primers:thesewouldinfactbeasetofprimerswhichhaveanumberofoptionsatseveralpositionsinthesequencesoastoallowannealingtoandamplificationofavarietyofrelatedsequences.Forexample,Compton(1990)describesusing14-merprimersetswith4and5degeneraciesasforwardandreverseprimers,respectively,fortheamplificationofglycoproteinB(gB)fromrelatedherpesviruses.Thereverseprimersequencewasasfollows:

TCGAATTCNCCYAAYTGNCCNT

whereY=T+C,andN=A+G+C+T,andthe8-base5"-terminalextensioncomprisesaEcoRIsite(underlined)andflankingspacertoensuretherestrictionenzymecancuttheproduct(theNewEnglandBiolabscataloguegivesagoodlistofwhichenzymesrequirehowlongaflankingsequenceinordertocutstubends).Degeneraciesobviouslyreducethespecificityoftheprimer(s),meaningmismatchopportunitiesaregreater,andbackgroundnoiseincreases;also,increaseddegeneracymeansconcentrationoftheindividualprimersdecreases;thus,greaterthan512-folddegeneracyshouldbeavoided.However,Ihaveusedprimerswithashighas256-and1024-folddegeneracyforthesuccessfulamplificationandsubsequentdirectsequencingofawiderangeofMastrevirusesagainstabackgroundofmaizegenomicDNA(RybickiandHughes,1990).

Primersequenceswerederivedfrommultiplesequencealignments;themismatchpositionswereusedas4-basedegeneraciesfortheprimers(shownasstars;5inFand4inR),asshownabove.Despitetheirdegeneracy,theprimerscouldbeusedtoamplifya250bpsequencefromvirusesdifferinginsequencebyasmuchas50%overthetargetsequence,and60%overall.TheycouldalsobeusedtoverysensitivelydetectthepresenceofMaizestreakvirusDNAagainstabackgroundofmaizegenomicDNA,atdilutionsaslowas1/10⁹infectedsap/healthysap(seebelow).

Somegroupsusedeoxyinosine(dI)atdegeneratepositionsratherthanusemixedoligos:thisbase-pairswithanyotherbase,effectivelygivingafour-folddegeneracyatanypostionintheoligowhereitispresent.Thislessensproblemstodowithdepletionofspecificsingleoligosinahighlydegeneratemixture,butmayresultintoohighadegeneracywherethereare4ormoredIsinanoligo.