实时定量PCR引物和探针设计操作步骤Primer Express软件.docx
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实时定量PCR引物和探针设计操作步骤Primer Express软件.docx
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实时定量PCR引物和探针设计操作步骤PrimerExpress软件
实时定量PCR引物和探针设计操作步骤PrimerExpress软件
PrimerExpress是实时定量PCR引物和探针设计的专用软件。
遵守以下三个原则有助于快速建立定量PCR反应体系:
1.所有扩增按照同样的原则设计(PrimerExpress);
2.所有PCR反应在ABIPRISM?
7000/7900上使用同样的热循环条件;
3.所有反应使用相同的PCR试剂。
引物和探针的设计原则
下述原则的重要程度由上往下越来越低,请尽量满足编号靠前的条件。
它们中有的已经在PrimerExpre软件中设置成缺省值,有的则需要在选择引物和探针时由设计者加以运用。
如果是设计SYBRGreen引物,也要选择TaqManPrimerandProbedesign并遵守这些规则,但是只需要合成引物就可以了。
TaqMan探针:
1.保持G-C含量在30-80%之间。
2.避免同一碱基重复过多。
特别是G,不可超过4个及以上。
3.5'end不能是G。
4.尽量使探针中的Cs多于Gs。
如果不能满足,则使用互补链上的探针。
5.对于单探针反应,用PrimerExpress?
软件计算出来的Tm值应当在68-70°C之间。
引物:
1.在探针确定以后再选择引物。
2.引物要尽可能地接近探针,但是不要重叠。
3.保持G-C含量在30-80%之间。
4.避免同一碱基重复过多。
特别是G,不可超过4个及以上。
5.用PrimerExpress?
软件计算出来的Tm值应当在58-60°C之间。
6.3'end的5个碱基中Gand/orC碱基的总数不能超过2个。
实时TaqMan引物和探针设计
BeginbyopeningPrimerExpressandselecting"File","New",and"TaqMan?
Primer&ProbeDesign".Thefollowingscreenwillappear.YoucanclosetheTaqMan?
Primer&ProbeDataboxasshown.
输入或插入序列
Importorpasteasequenceintothewindow(Importshown).TopasteasequencefromaWordortextfile,firstcopyittotheclipboard.Besuretoonlyselectthesequence(includingnumbersorannotationsisOK);donotincludeextraneousinformationsuchasaccessionnumbersetc.Next,select"Edit"and"Paste".ThesequencewillappearintheSequencescreenofPrimerExpress.Or,toImportaSequence,clickthe"ImportDNAFile"buttonasshown.Thesoftwarewillthenaskyoutolocatethesequencefile.Selectitfromafolder,harddrive,disk,ordesktop.Again,noannotationsshouldbepresentinthissequence.
Afileisthenimportedafterselectingthefilelocation.
保存输入的序列
Select"File"and"Save"togivethesequenceaname.ThiswillbedisplayedintheFileNameBoxandwillsavethesequenceintheArchiveFolder.
引物和探针设计参数
Clickthe"Parameters"tab.ThisdisplaystheUniversaldefaultparametersusedtosearchforsuitableTaqMan?
primer&probesetsforreal-timeassays.Itisstronglyrecommendedthatyoudonotadjustanyoftheparameters.
引物和探针的排序及选择
PrimerExpressisnowreadytofindPrimersandProbes.Clickthe"Primers"tab,select"Options"and"FindPrimers/ProbesNow".Thesoftwarewilldisplaytheprogressinthesmallwindowbelowthesequence.
**Pleasedisregardthe"OptimalPrimerPairsOnly"checkboxandthe"Penalty"heading.BycheckingtheOptimalPrimerPairsOnlybox,youwillbeseverelylimitingtherangeofyoursearch,sincetheparametersitemploysarenotbasedonTaqMan?
designguidelines.ThePenaltyscoreassignedtoyourPrimer&Probesetisbasedonfactorssuchasampliconlength.SincethedefaultTaqMan?
designparameterskeepampliconsunder150bp,thiscanbedisregardedaswell.
Primer/probesetswillbelistedwhenthesearchiscomplete.ScrolltotherighttoviewtheProbes.Clickonthe"Start"headingunderprobestosortprobesbysequence.Thiswillgroupsimilarprobes,simplifyingthesearch.
探针的选择
Selectaprobethatislessthan30bpinlengthandcontainsmoreC'sthanG's.Theprobesdisplayedareonthesensestrandonly.IftheprobesdisplayeddonothavemoreC'sthanG's,thenyouwillneedtousethecomplementprobe(asillustratedinthisexample).Ifyouneedtousethecomplement,makesurethattheprobeselectedheredoesnothaveaCatthe3'endoftheprobe(otherwise,thecomplementwillhaveaGatthe5'end?
whichisnotallowed).
Theprobeselectedmeetsthefirstcriteriaabove,butnotthesecond(9G's,5C's).Highlightthisprobe.
Returntothesequencebyclickingthe"Sequence"tab.
LockintheprobesequencebyclickingtheProbeButtonontheToolBarandhighlighttheprobesequence.Theprobewillturngreenandbedisplayedinlowercasewhenitislocked.
引物选择
Findcompatibleprimersbyreturningtothe"Primers"tab,selecting"Options"and"FindPrimers&ProbesNow".Thiswillfindnewprimersetsthatwillworkwiththeprobeyouhaveselected.Youcanclickon"Start"underForwardPrimertosortthedisplayedsequences.
Searchforaprimerfromthelistdisplayedthemeetsthefollowingcriteria:
1.Nomorethan2G'sand/orC'swithinthelast5basesonthe3'endoftheprimer;and
2.Norunsofidenticalnucleotides,especially4ormoreG's.
Fromthelistofforwardprimersdisplayed,selectaprimerthathasnomorethan2G'sand/orC'swithinthelast5basesonthe3'endoftheprimer.Highlightoneoftheprimersthatmatchesthiscriteria.Ifnoforwardprimermatchesthiscriteriathenselectaprimerwith3G'sand/orC's.Theexampleshownbelowmatchesthecriteriaandwillserveasasuitableforwardprimer.Onceyouhaveselectedtheappropriateprimerclickonthe"Sequence"tabtoreturntotheSequencewindow.
Locktheforwardprimerbyclickingthe"ForwardPrimer"buttononthetoolbar,thenhighlightingtheforwardprimersequence.Abluearrowwillbedisplayedundertheforwardprimershowingthatitislocked.
Clickonthe"Primers"tabandperformanewsearch.ScrolltotheReversePrimersdisplayedandselectareverseprimerfollowingthesamecriteriaforforwardprimerselection(G/Cruleonthe3'endofprimer).
ReturntotheSequencepageandlockinontheReversePrimerusingtheReversePrimerTool.
Thisnowdisplaystheprimersandprobeyouhaveselected.ReturntothePrimerstabandperformonefinalsearchtodisplayyourresults.
保存搜索结果
Clickon"SaveList"atthebottomofthescreentosaveyourselectioninatabdelimitedformat.Click"Order"togenerateaneditable/printabletextfileofyoursequences:
互补探针的选择
Intheexampleabove,youmustusethecomplementaryprobesoastoinsurethattheprobehasmoreC'sthanG's.Remember,theprobeyouusecannothaveaGatthe5'end,thusthesenseprobeusedforthissearchcannothaveaCatthe3'end.
Inordertogeneratetheprobecomplement,returntotheSequencescreen.Highlighttheprobesequence,select"Edit",and"CopyComplement".Youwillnotseethecomplementarysequenceatthispoint;itiscopiedtotheclipboard:
ReturntotheOrderwindowand"Paste"thecomplementinthiswindow,overwritingtheprobedisplayed.Youhavetheoptionofeditingtheprimer/probenames,andaddingthereporter/quencherdyestotheprobesequence.
ThisdocumentcannowbesavedandputintoaWorddocumentorattachedtoane-mailmessage.
在ResultsArchive中保存搜索结果
YoursearchcanalsobesavedintheResultsArchiveFolder.Clickonthe"Results"tab.
Theforwardandreverseprimersaredisplayedintheirrespectiveboxes,andtheprobesequenceisdisplayedinthe"CycleParams"boxTheprobesequencedisplayedistheoriginalstrand.Toview/savethecomplementarystrand,highlighttheprobefromtheSequenceandselect"CopyComplement"."Paste"thecomplementprobeintothe"CycleParams".Thecomplementaryprobestrandisnowdisplayed.ItisimportanttonotethatifyouleavetheResultspage,theprobesequencewilldefaultbacktotheoriginal.EachtimeyoureturntotheResultspageyouwillneedtore-pastethecomplementaryprobestrand.Note:
TheinformationdisplayedbelowtheselectedprimerandprobesequencesshouldbeignoredwhenperformingTaqManAssays.TheUniversalTaqMan?
Guidelinesdonotrequireyoutoperformoptimizations,thus,thecycling/concentration,etc.informationdisplayedherecanbeignored.SavetheResultsbyselecting"SaveResults".Amessagewilldisplayshowingtheresultsweresaved.
打印结果ToprinttheResults,select"OpenResults"fromthe"File"menu.Thelast(newest)resultsfilewillbethelastoneinthelist(atthebottomofthelist):
Highlightandclick"Open".
Thisistherelevantinformationneededtoorderyourprimer/probeset.Toprint,clickanddrag,highlightingtheinformationyouwantandselecting"Copy"fromthe"Edit"menu,placingitontheclipboard.ThisshouldbeeverythingfromtheSequencenamethroughtheTaqMan?
probeannealinginformation.
Thisistherelevantinformationneededtoorderyourprimer/probeset.Toprint,clickanddrag,highlightingtheinformationyouwantandselecting"Copy"fromthe"Edit"menu,placingitontheclipboard.ThisshouldbeeverythingfromtheSequencenamethroughtheTaqMan?
probeannealinginformation.
YoucanthenpasteyoursequenceinformationintoaWorddocument;fromhereyoucanprintacopyforyourrecords.
订购信息
Besuretoincludeinformationonyourneededsynthesisscaleandthecorrespondingpartnumber,yourreporterdye(s),yourquencher(TAMRA),andyourpersonalinformation(name,institution,address,phonefaxetc.).
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