实时定量PCR引物和探针设计操作步骤PrimerExpress软件

1、实时定量PCF§|物和探针设计操作步骤 Primer Express软件Primer Express是实时定量PCF§|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PCRK应体系：1. 所有扩增按照同样的原则设计(Primer Express);2. 所有PC版应在ABI PRISM ?7000/7900上使用同样的热循环条件；3. 所有反应使用相同的PCFF式剂。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

2、SYBRGreenUl物，也要选择 TaqMan Primer and Probe design 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 G4. 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Primer Express?软件计算出来的Tm值应当在68-70 0 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.

3、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Primer Express?软件计算出来的Tm值应当在58-60 0 C之间。6. 3' end 的5个碱基中G and/or C碱基的总数不能超过2个。实时TaqMan引物和探针设计Begin by opening Primer Express and selecting "File", "New", and "TaqMan? Primer & Probe Design". The following screen will appear. You ca

4、n close the TaqMan? Primer & Probe Data box as shown.输入或插入序列Import or paste a sequence into the window (Import shown). To paste a sequence from a Wordor text file, first copy it to the clipboard. Be sure to only select the sequence (includingnumbers or annotations is OK); do not include extraneo

5、us information such as accessionnumbers etc. Next, select "Edit" and "Paste". The sequence will appear in the Sequence screen of Primer Express. Or, to Import a Sequence, click the "Import DNA File" button as shown.The software will then ask you to locate the sequence f

6、ile. Select it from a folder, hard drive, disk, or desktop. Again, no annotations should be present in this sequence.A file is then imported after selecting the file location.I n(I Man Probe * i*诉HE1珈g，知t*I3¥DI>|Ihm*l«J 呻 nM 昨中 1AACTTrCTG 6妃GZkMTEAA GUTtC心：TACijCCCCTiATCi/lGCfiGW. UTTC1

7、KGG TGCCGKTR AUWATne 婉ig3 场口WAtrn QATAKGCAA WnTAfiTT TCTATTtCAT 'TTKA2CiR，KGARTCGG TCXCAGCGG U.r4jTGTTTC MT皿更iTAATKTAT31 gta£Ti Gtimw fMATg KITH克ATT WT3牝炬 TAEWsH TMCGCCATC TfG统心TG QAUCTiACG KGCAJCGAn utattatac tatttttw 葡町心TTLi iMCTtigM 虹TMVHGT aK心岖 CtAATIGAGG GAMTTCGfA AGAAATTTAA 心技怡gAMiCCC

8、wG T碱UwT GCGWKBAGT CATCflGATCA ATrrcrmt amtitgcaa TKiGATAKiT ATTTKiTCrAT&ACTTCnGG AArATTT&M CflCTWGTXTT WnCCMiT KTMT3TA TKA7TC&H GKGMCm (XAMTtXAT AnCGTWCr OTTBCTIM CXtGfiXCG <TTCGMM 硬缺 KGAKWTTAGTKntrr nagatggct TAA原心AT TTGF岖:曲 CaMTATTT C7CCG0GTTT MjTuT职C<X AT量尹顷a ALC&OCAG ATCIU

9、iCCM OXGTK心间TTAACm 4TTKAA顺 T(iCTOTCW£ise 闽臼 ?5& 观35& 垂狎 45& 瑚 550潮75OCMTArKA ATKMTG&T 手心壬度 WTKG，'吹保存输入的序列Select "File" and "Save" to give the sequence a name. This will be displayed in the File Name Box and will save the sequence in the Archive Folder.lac

10、iMnn >1ffyiwg Cnnid T Prtwr二m T ftrtziiw顽松“1"|!i r hitef：ni.c-Eaccito-rsiA gcjulmtcm caaezc.： tjexcclci-a55sMCO 花找 TA-FTCITKA£*OTTTC 岫国CM wotwcaa itmxnnjcynr KCArcrock5 I3W”.“We4 ,一 I 一 .immiUHI TMCnmE MrArnUM AGrwjcrrr g 堀tgt aitcccMjT KTATOAT FCFAAirATA TKATKCKi mr侦ce 匚心we cfiwaKfiATU

11、jC&WI匚LA 岫 TTMtTLA AmtMiWG IBCTtTCCM： CXXLMiMj TMTACCiWC GCGnnaic 心mmn RcnTtz CaTCTCTI A£i4AAA£A CBOOnn MDJTAUC G7GIETTUUTTMiimnc AxcnjAirrv ctocthkw jutwjuct qiwocaa GThChHCAT WTTTTGCttJ 砌CAT:LA CCC昭ACW XTTLGKjU TTCTTCWT TGF职;就 FESMi gCCH心 CCWCCTT TTK£<MAT TK心MH! gCT0K MHGTtH

12、IL心TWT TTGCACtATT TCUnm THTTTAAA fMKJUGM TTCUAKit AfiAAAiTTM CKTXMA AGAATTOT CtftfiATZrT CXC&AffTTT ，侦CAUTAJX WCGCC>tfj TMTAJgT 吒HMCt匚 ATfltT的岷 St由蹒CT CATtiTCA HCGKT：垢踩谜3 WArreg atkxtttk awittgcx 4JGiWTTTM T&GtTAlTGT JJTTnTC 心五3上 Wg：g 占 mauanc ATCGMAOi AGHTCIOGr TTCHCCTtt MTOCty CWOTaA n&

13、#171;TATdW> TKMTCTMC GCT«aCGjMj COOTTTOi CAIGCGCCGT JG£4£A：UKiAlTaAi 2A；A；rflXE二CV.ATQyTlilTATTAt CtXCtfTtjCC Client J 1 rfttrtjTrTxka/afgnAA融 lAMAAATT octkat 虹C AnWATiiAT W.4UTJ TT&mAT mrmw atke .roaCT “EAT曲cr.rTTT. ZTAKTZAT叮 CCfiTOGX.'T FfSCA厂！引物和探针设计参数Click the "Par

14、ameters" tab. This displays the Universal default parameters used to search for suitable TaqMan? primer & probe sets for real-time assays. It is strongly recommended that you do not adjust any of the parameters.引物和探针的排序及选择Primer Express is now ready to find Primers and Probes. Click the &qu

15、ot;Primers" tab, select"Options" and "Find Primers/Probes Now". The software will display the progress in the small window below the sequence.* Please disregard the "Optimal Primer Pairs Only" checkbox and the "Penalty" heading. By checking the Optimal Pr

16、imer Pairs Only box, you will be severely limiting the range of your search, since the parameters it employs are not based on TaqMan? design guidelines. The Penalty score assigned to your Primer & Probe set is based on factors such as amplicon length. Since the default TaqMan? design parameters

17、keep amplicons under 150 bp, this can be disregarded as well.Primer/probe sets will be listed when the search is complete. Scroll to the right to viewthe Probes. Click on the "Start" heading under probes to sort probes by sequence. This will group similar probes, simplifying the search.探针的

18、选择Select a probe that is less than 30 bp in length and contains more C's than G's.The probesdisplayed are on the sense strand only. If the probes displayed do not have more C's thanG's, then you will need to use the complement probe (as illustrated in this example). If you need to us

19、e the complement, make sure that the probe selected here does not have a C at the 3' end of the probe (otherwise, the complement will have a G at the 5' end ? whichis not allowed).The probe selected meets the first criteria above, but not the second (9 G's, 5 C's). Highlight this pro

20、be.口faqMan Prob&#1. S加Permm零 Y Rxn OwkI T Prhn噌rs I M叩 T Bacl。皆】Re$ult8 iiMFT14HProbtPrimerGt"，TfhWGCStartT AATGAT MQC ATTCGTGTGC21020&965TTCG ATSTCGCTGGfC AGCS254T AA7GAT ATGC ATTCGTGTGCg196974CGATGTCWTOGCCZJiCCG£54TCGTOVaCAGCATGTT2U2Q6370ATGTCGaTGQCC AOCOQOAC湖KGTDTOCSCATOTT21+206

21、670妇 GT C6GTGGCC AGCGGO ACZ3TCGTSrQCSQATCTTg1?就74TCTCQGTGCGCGSGGC沔TDGTGTGCAGCATGTT±1519&&74TGTCGGTGGCC A6CGG G AC3nrTGCAGCATGTTCG2t519G3747GTCCGTTCCC AGCGG G AC256jTGrGCAGCAT&TICG21519哭74TQTC&tjTGCCC AGC&GGAC路8jTQTQCAGCArGTTCGm196S网TOTctaroccc皿:TtTfiCAOCAmTM215i 96&74TQT

22、C60TMCC264LTGTGCAGCAFGnCG2151勺舶74TCTCGGTGCCC ACCGGG AC2UjTG TGC A GCATGnOG215196S74TCrCGGTGGCCAGC0G0此264jIGTGCAGCATGITCG2t5196874TOTCGGTGCCCGCGG "C264F&TGCAGCATGTTCG21519&&74TGTCGGTMCC AGCGG G AC2SSF&TGCAaCATGTTC&2tS19G874TOTCtGTGGCC ADCQGGACrGTOCMOTWTCG215196874TOTCGOTQOCC

23、AGCOGOAC双rGTCCWjCriTGTTCSg19曲74TGTCGGTGCCC 由 GC 廊 C AC264FGTGCAGCATGTTCG21519&s74TGTCGGTG15CC AGCGG G AC264rGTGC*6CATGTTCt32t5196&74TGTCCGTGCCC AGCSG G AC264lOTOCAGCATOTTCt?215196874TGTCtSDTWCC AGCQO G AC264H：GtOtGCAGCATDTT21?197078TgtHG6Cl：旨 GCOGMg药BjTGTGCAGCftTQTTCG317i e7079TCTGi：<CA&a

24、mp;CCO3ACC3TCrGCA6CATGTTCG27197078TCGG7GGCC AGC6GG ACG259jTGTIjCAGCATGTTCG217187078TCGGTGSCC AGCGGQ ACO矗4Return to the sequence by clicking the "Sequence" tab.Lock in the probe sequence by clicking the Probe Button on the Tool Bar and highlight the probe sequence. The probe will turn green

25、 and be displayed in lower case whenitr， L： TaqMan Pr-obc -*1H B :画U.一kAW FTLF归ci r-i.LiRcr Rttwf小4frnwRTGTTT口二牢-i-rm- 寸&dfmnrLin#San siSktrn玉口nitenc寸 Fmramg TRxnccijiiT Primwg 丫 Mwp Tiecip色 丫 Fib N>mt |A3 Hlp L*ft9lh 300 bp,Stkction 拓9 皿 39 Doubtt Strjndedtili lLJdl. ll .lllljtao.JblllL Ll A

26、JL 1. X J_ I. IL 1 L 1 1 11 k j 1 I L I b u . 1 I j I TI i 1. u J L I L I L I 1 i uCCC MMGQK X/h(孕 叫二 了虹m 罚ATGAGCACG4 TATTCTTCG6 TGCtCTGTTT TGACTTCTO WAITTGAA 物ACAAGATTTC WBTTGGACAA AGTAMCTIT OGTMGTGT WTCCCWGT 15® gWTOtAA prTTTAGTT TtTAntCAT TETAAlfiATA TpCATTCGT 290 羿40加皿m 与乔Eg匚gg邮日戚GM亩如顽j 0 f

27、oaiLI l|fC AATUftTOUA CnjCTATCAT ATKGTAACT GCtHUCOA 观 I tfTTATAACAT (j(jTTTTtjC(Xi GWAKCC CCCAGAZ1CC6 MTTCBGflA 350KTTTGGATT TGrTTAAGAG TATTTMiAAC fiACM&TG曲1 CCMOCMTT 做 IGGCGCCATC TCGCAAATG GWCTAflCG 心口TCTKH TCAGATGGCT 45ft TrfifATfiATT TfTATTiT TATTTTAAA T皿成而 TTGTT戚蜡 5引物选择is locked.sequences.Fi

28、nd compatible primers by returning to the "Primers" tab, selecting "Options" and "Find Primers & Probes Now". This will find new primer sets that will work with the probe you have selected. You can click on "Start" under Forward Primer to sort the displaye

29、dSearch for a primer from the list displayed the meets the following criteria:1. No more than 2 G's and/or C's within the last 5 bases on the 3' end of the primer; and2. No runs of identical nucleotides, especially 4 or more G's.From the list of forward primers displayed, select a pr

30、imer that has no more than 2 G's and/or C's within the last 5 bases on the 3' end of the primer. Highlight one of the that matches this criteria. If no forward primer matches this criteriathen selectwith 3 G's and/or C's. The example shown below matches the criteria and will serv

31、e as a suitable forward primer. Once you have selected the appropriate primer click on the "Sequence" tab to return to the Sequence window. -r IciqMdn Probe-#1 ,日SequgrjEFT P.ram言 T Ibcn Cond YPrimers Mau 丫 Recip、Forward Pr imerT岫Man I与UMLengthTmxccPrimerLengthTms«c1%206030TCG h=TGCAd

32、CA7CTTCGAT217187078rc19g55TCGTGTGCA(3CA7OTTCGft2*7187078TC1953TTWTC-TOCCAT&TTCG.51?IB79TC2050TCGTGT GC A3C ATOTTCGAT2t7IBTO79TCIH1955TCXT&TGCAaCATOTTCG21713TO78rd14河MSOATTCGTCIGCACCATGTTCfi217IBTO73TC?nsnTriTATmArlir： ATATTHTIAT2K71RTO7ALock the forward primer by clicking the "Forward P

33、rimer" button on the toolbar, then highlighting the forward primer sequence. A blue arrow will be displayed under the primer showing that it is locked.primersa primerforwardSequence Paramsnf RKn CoiiFl Primer胃Mag 陆丫 R巳suns )L J 2OT to 207" oub It StrAndHl50100150LtfigWi JL 11380bp5AOrTTCC

34、GicCGAG'nGA GCACAATCAA GAATCCAAAC TAJCACCCCTA *1TWC«A T&TKTTCGG TGCCGKm TGMTTCTGG AATATTTGAA 就坂AEC AAGTCGACAA AGIMACTTT CAGTAZCTGT AATCCCWJ GATATCGCM T&TEAGTT TCIATTCCAT TTTAaTGaTA TCCAfTGTTCCAJGCATGT兰伊土ugg 七ggmicgg godAfiCGTA GGAAATjOATI冬巨画EL MKiATCTM CTKTAKAT ATXGTAKT GCGKCK妙 mSwC

35、AT GGTTTTGCGG GAAGATCCCA CCCAGAACCG ACTTCMjAA 1CTTTGGATT TGTTCAAGAG TAITTMAAE AAC颇TGAA CCGAGCAATT TOCGCCATCGWLTAACGAGTTjTTCTTTtAGATQGCTClick on the "Primers" tab and perform a new search. Scroll to the Reverse Primers displayed and select a reverse primer following the same criteria for fo

36、rward primer selection (G/C rule on the 3' end of primer).TaqMian Probe # I -三可日零 T R善n Cond T Prim噌辑 Ma口】 R律cip电 f Results 旧既*”鼻PrimerPrubStartLnthTni9GCPr iitirlengthjGCCAGCGGGACG25623羽C ACTG 通 TCCKTTTC C7 ACG CTC：i(?(XflGCGG&ACf'2572460箱AMCTGMTCX ATTTCCTACGCTC&2>«C*GCGGaAC

37、C±56相4耳A AC ACT6 a aTCC T T CC T M 就知心 GGGMG2592A5942AA A were AATCCATTTCCTA.CW：W3&££ AGCGGGACG264245S用C ATT6 A A AC ACTS AATC CTTIIX&9jGOCASCa?(?ACCZ64255?%Q rtTTG & A ACACTC fATC C ATTTCCT633GCCAJGCCGGACG2&4265935C ATTG A A ACACTGAATC C ATTTCCT A693GCCASJCGGGACG26427

38、60nC ATTG&A AC ACTG ATC C TTTCC T AC&9，&心成的*e26沸35GTTACM AHGAAACACTQAATCC A7638CMKCGG 心27127襄G TT AC 冉 TU AFTCrtAACA CIG A A ICC A TrejWAGCGGGACC2712&59KGTTACATCaFTCaAAC ACTGATCCAT'7£:GCCWXGGGACC272275933AGTT AC A TCAT TGAAfi.C ACTGA ATCC A77jMXAGCOCQACC27228的32务 GTT AC A TC

39、AT TGAAC ACTS* ATCC A?7网皿 林四序AM洒2446就 ATTAQrtAT AT&rtT AQC101?WCAGCCOaACG25句MAg心亡 AGtl ACG焉ATA70AJAOC102j&rcAGCooaAtt理355S44G 耍 MGC AGTTACGAAT ATG AT A6ID3戏尤MCGOgW纫235?侣T GTOC yCM T T 枷 A AT 内 m104jMAGCGGOACC246042TGA0CM?GCAC7TrtCGAATAT0AT104Return to the Sequence page and lock in on the Rev

40、erse Primer using the Reverse Primer Tool.TaqMan ProbeIMoquoncGT Parmnns Thkh CondT Map 丫 Raclg 丫 机睥ults f il Name DJ)PHBF§fc3 qb 一 吹吵塑1 比里 1'LwHi 13B0 bp.Stk-dhn31】上字3f 1 O Ootjlsl* StrvwMCTTCCTG GAfWf GEMMT心 GiATTCAMT TCCCTA 53ATGACCW5GA UTTCTTCGG TGCCGTGTTT TGACTTCTO WATTTGM l&ftACAA

41、GATTTC AAGTGGiCM AGTAAACTTT CAGTAAC7GT MTCCCAAGT 150GATATCGCM UTTTTAGTT TCTATTLCAT TCTMTGATA TTjCAlfTCGT W&&fTGCMCATCT TCGATGttgg t通匚Eqcgg 卵国ACCGTA GdAAATGGAT 253IMTGTTT MTGATGTM CTtjCTATCAT ATTCGTMCT GCGTGCltM 306QHATMUT GGTTTTGCGG GMGAXCCA CCCAGAACCG AtTItGAGM 350TcrrnxATT tgfxaa&ag ta

42、tttggax aataggtgaa cc(iaccaatt 娅KfiCGCCATC TCGXAAATG GMCTAACG AGnGHtTT TgTGGCT 4WThis now displays the primers and probe you have selected. Return to the Primers tab and perform one final search to display your results.口 raqLten Prob臼毒1Y&口 舀Suqu口口匚已* Params Rkh 仁口门曰 TpriniT5 丫 Map RecipiE Result

43、zs r«rwAr4 Pr im*rkqfm Pm4Start Lanfth Tn WOCPrlntFStart Lenflfc T *OC1M 2tl *030 rCGTCTtSC AGCATGTTCGT2171 宕TO 76 TCMTG保存搜索结果Click on "Save List" at the bottom of the screen to save your selection in a tab delimitedformat. Click "Order" to generate an editable/printable tex

44、t file of your sequences:互补探针的选择In the example above, you must use the complementary probe so as to insure that the probe has more C's than G's. Remember, the probe you use cannot have a G at the 5' end, thus the sense probe used for this search cannot have a C at the 3' end.In order

45、 to generate the probe complement, return to the Sequence screen. Highlight the probe sequence, select "Edit", and "Copy Complement". You will not see the complementary sequence at this point; it is copied to the clipboard:7>.l;LdL少-Jrt'-htFroiit* IRnr-ari FTTit?r" ”

46、r11 H v - T-TF嗯* I、HfiratmH*«TemornLin?e±5心n S：1!«laqMen Probe *1S E3soquonc口Kim口Y gp 丫 REUipg 用Mgr F.IMIwtj- 煦盹岫榷岖I曲Length1390»r 岫 IS eke kem217234 EJ boubie Stranded I I li I L I L 1 L lT.I LJIj.npMrKCTG GMG场兀耳成心N攻 GMTCCAW Mt虹心L TATTCrnCGG 戚任花口！皿:T7CTGG ACJWGATTT AAfiTCGATM AlG

47、TWUCTTT CMHWT mrmGCM tett殂itt tctatttcai thaa何mGGWTIGgTTACACCCCTA W町TTL灿TC0TOC阳HIUT杠如 t-ggeepq 匚 g g_g迎pGCGTAaatcaTjTaa曲*ct saniGCTtMKAGTGTTTCGHATAJCAT RTTTCGAn TOCGmiT TLGCXGAnGTTTFLiCLL TGnCMAG K网心尚TCTATIWCGAAGATCCti TATTTCGAJUC GAAATTAACG TATTTTTMA *一心TTG7心炒皿G性TTC泌皿 AtfGGTW CCtiXTAATT AGTHsTlCTT

48、'KMiATTiGCT TAA3CAAGAT TTOrTftGCAG CGGAATiTTT CKCOfiaUT353 蜘 4Mthe complement in this window, overwriting the probeReturn to the Order window and "Paste"displayed. You have the option of editing the primer/probe names, and adding the reporter/quencher dyes to the probe sequence.This do

49、cument can now be saved and put into a Word document or attached to an e-mail message. to the Results page you will need to re-paste the complementary probe strand. Note: The information displayed below the selected primer and probe sequences should be ignored when performing TaqMan Assays. The Univ

50、ersal TaqMan? Guidelines do not require you to perform optimizations, thus, the cycling/concentration, etc. information displayed here can be ignored. Save the Results by selecting "Save Results". A message will display showing the results were saved.TaqMan Prahwi OrderE0PIqqe臼 srnthesizb

51、the fntle/ang olLgos For ffie end bi 11 try £<Hee ;equEics:CRWePSA3.gb-196FTCGTIGTiCAGCATIGnCGATI *<|MPro卜、E 将协"to79_TuZTClHome;equenc 己；DRO<jMaP5A3.gb-«FRMC A£GC AGTTMGWT虾GHTM 匚and the Ttj帕rt pnjbE；Hee :DROGW0PSA?. gh- 217TSequence： F Ab I- CGTCCCGC TOCC ACtGA-R AIhonks

52、 veryusernJn在Results Archive中保存搜索结果Your search can also be saved in the Results Archive Folder. Click on the "Results" tab.The forward and reverse primers are displayed in their respective boxes, and the probe sequence is displayed in the "Cycle Params" box The probe sequence displayed is the original strand. To view/save the complementary strand, highlight the probe from the Sequence and select "Copy Complement&q