一、单重Taqman引物探针设计:(为了确保引物和探针的特异性,最好将设计好的序列在www.ncbi.nlm.nih/blast中核实一次)1、探针:探针的设计应该在引物的设计之前①长度:18~35(18~30之间最好、最常见),最长37;太长淬灭效果不好。②TM值:PrimerExpress软件计算出来的Tm值在66~72℃之间(最常见68~70℃),最好为70℃,确保探针的Tm值要比引物的Tm值高出5~10℃,GC含量在30~80%(最好40%~70%),因此探针最好是富含GC的保守片段;(PrimerExpress:DonotexpandtheTmrangebymorethan2°Cfromthedefaultrange)。③探针位置:尽可能地靠近上游引物,上游引物的3'端离探针的5'端为1-20bp(一般10个以内),最好是探针的5'端离上游引物的3'有1个碱基。④5'端应避免使用碱基G,5'G会有淬灭作用,即使被切割下来这种淬灭作用也还会存在;如果选择FAM-dye在5'端第二个序列也不能为G(Ginthesecondpositiononthe5'endinFAMdye-labeledprobescanreducefluorescentnormalizedreportersignal)。⑤可选:整条探针中,碱基C的含量要明显高于G的含量,G的含量多于C会降低反应效率,这时就应选择配对的另一条链作为探针。要同时考虑在正反两条链上设计引物与探针。若找不到完全保守的片段,也只能选取有一个碱基不同的片段,且这个不同的碱基最好在探针的中间,且最好为A或T。⑥Repeatingoligonucleotides:a.避免探针中同一碱基重复过多,尤其是要避免4个或超过4个的G碱基出现,即≦3(Avoidrunsofidenticalnucleotides.Ifrepeatsarepresent,theremustbefewerthanfourconsecutiveGresidues.);b.避免连续的6个A出现(ConsecutiveAresidues:AvoidsixconsecutiveAresiduesanywhereintheprobe.ConsecutiveAresiduescancauseahighNoTemplateControl(NTC)signal);c.避免探针的中间区域含有2个或以上的CCdinucleotides(AvoidtwoormoreCCdinucleotidesinthemiddleoftheprobe,whichcansometimesreducesignal)。⑦检测探针的DNA折叠和二级结构(最大连续的碱基配对数=4:Maximumnumberofconsecutivebasepairingsallowedinaprimer=4;最大的总的碱基配对数=8:Maximumnumberofbasepairingsallowedinaprimer=8):a.避免自身形成环状发卡结构(HairpinLoops:IfaDNAstrandpossessesaself-complementarysequence,itmayformasecondarystructure,oftencalledahairpinloop.Whendesigningprimersorprobes,avoidregionsthattendtoformsecondarystructures.Ifsecondarystructuresarepresent,primersorprobesmustcompeteagainstthecomplementarystrandforbindingtothetargetsequence,whichreducesPCRefficiency.),b.避免自身二聚体形成(Self-Dimers:Primersorprobesthatpossess3´complementaritywiththemselvesinthePCRreactionmayformanothertypeofnonspecificproduct,aself-dimer.NonspecificproductformationdecreasesthequantityofspecificproductproducedandmayaddambiguitytothePCRresults.ThePrimerExpress®softwarecalculatesandeliminatesfromconsiderationmostoftheprimersandprobesthatareself-complementaryorpossessstrongself-dimertendencies.),c.避免交叉二聚体形成(Cross-Dimers:Primersorprobesthatpossess3´complementaritywiththemselvesoranotherprimerorprobeinthePCRreactionmayformanothertypeofnonspecificproduct,across-dimer.NonspecificproductformationdecreasesthequantityofspecificproductproducedandmayaddambiguitytothePCRresults.ThePrimerExpresssoftware®calculatesandeliminatesfromconsiderationmostoftheprimersandprobesthatareself-complementaryorpossessstrongdimertendencies.)。⑧可选:3'端的淬灭基团也可以标记在探针的内部核酸上。5'端染料最好连接在T或C碱基上。⑨可选:探针应高度纯化(HPLC);探针储存液应为100um浓度,工作液为10um浓度并分装25ul/份。⑩反应体系中探针浓度的优化主要依靠背景信号强度、探针与靶的结合效率、PCR扩增的效率、引物的浓度等进行优化。一般探针浓度在0.1~0.4uM范围内进行优化。若用新合成的探针进行优...