1、 Primer Express Operation Guide Primer Express v3.0 Primers and Probe Design For Real-Time PCR Primer Express Operation Guide Primers/Probes Design Guideline TaqMan Probe Primer Probe PrimerY w, PCR *JD 50-150 bp 3 G/C % 30-80 % 5 Y $dI5 4Hj GY $ Tm= : 68-70(Quantification assay) 65-67(Allelic Discr
2、imination assay) Tm= : 58-60Probe : 1325 bases (TaqMan MGB probe) 1330 bases (TaqMan probe) Primer : 20 bases (Optimal) 5 6H AY $ 5L KSH G (o FAM-dye 5L KZHp G) oC GYstrand *probe b5 3LY 4H 3Hj G (GGG-MGB-3 or GGAG-MGB-3)a5probe Y 9 2HjYCC di-nucleotides a3LYH t 2H C+Ga: TaqMan MGB probe b: o Primer
3、 Express Operation Guide Primers & Probes for Quantification Automatically Design Primer Express 3.0 File Newo “TaqMan MGB Quantification TaqMan Quantification” OK Tools 2 “Add DNA File” d 0_d2h ”Add”d Z dcfu ”Sequence” Tab4 Copy & Paste7 h if * Primer Express Software% a .dan, .txt, .ab1, .abiY , +
4、 Z Y qC (J database downloadh , ) Primer Express Operation Guide o ” Double Strand “, C6j double stranded DNA sequence ToolsjYo 1. Exclude:Y ”Exclude Selected Bases” Z - o_Y do “Exclude”dC 9 Primer Express Operation Guide 2. Junction: 8jjunction ”Junction” - o juctionY ( 2 bases)do “Junction”dC 9 z
5、f 3. ”Parameters” TabQ Primer/Probe Tm= I Primers/Probe Primer Express Operation Guide Q ”Sequence” TabdTools Find Primers/Probes d C 0 G Y Primers/Probe pairs Primer Express 6Q Candidate Primers & Probe pairsdS search Q 50G d , ”Primers / Probes” Tabf ”Location” primers & probesY djY dh f ”Sequence
6、” Tab6j ”Primers / Probes” TabYS Primers/Probe: j lJProbeYd Forward Primerd l Reverse Primerdhjf (*(j 3Y ) Primer Express Operation Guide ”Primers / Probes” Tab6 Z Primers/ProbeY d ? o G Y d? o1 K 2+ primer/ probe design guidelinef MGB ProbeY o du ”Parameters” Tab o ”C ” ” G ”Y* SoYf primer/ probe s
7、et dY File Save Asf ZoYprimer/ probe set d Export Order InfoY d k 50H Primers/Probes do Export Primers/Probes ListdjG .txtd Excel f Primer Express Operation Guide Manually Design , 0Q Primer/Probe d Primer/Probe Y SH-Y Quantification Documentd( Y (k K 3+ )f 1. ProbeY : ”Sequence” Tab Z Probekhighlig
8、ht ( 25 basesd1 )d Edit Copy (Ctrl+C) d1h Tools Primer Probe Test Tool do Y document type (C “TaqMan MGB Quantification TaqMan Quantification”)d( Parameter ”Default”d Paste (Ctrl+V) Z h Probe 1 d C H Tm, %GCqJIGf W : probe KSH Gd& CYf G Tm t = (68to 70)d Probe 1oYld(HY Tm, %GCqd G Y Probe (k primer/
9、 probe design guideline)do QGYldSh ”Trim”d 1 ZJoY dhY Probef Primer Express Operation Guide ProbedQ ”Sequence” Tab Z Probehighlight ( Edit Annotate ” Probe” Z probex d probeY6 z f d Tools Find Primers/Probes d Probe Y Forward / Reverse Primersf $ 0 ,Q Y Primers1h7 Forward / Reverse PrimersY f Primer
10、 Express Operation Guide 2. Forward Primer : ”Sequence” Tab Zk Primerhighlight ( 25 basesd Probe )d Edit Copy (Ctrl+C) d1h Tools Primer Probe Test Toold Paste (Ctrl+V) Z h Fwd Primerd CH Tm, %GCqJIGf Forward PrimerY Tm= 58-60 d Tm= Ld Fwd PrimeroYl d( H Y Tm, %GCqd G Y Primer (k primer/ probe design
11、 guideline)doQGYldSh ”Trim”d 1 ZJoY d%hYf Fwd Primer d Q ”Sequence” Tab Zhighlight d ” Forward Primer” x dY Fwd Primer6 z f Primer Express Operation Guide 3. Reverse Primer ”Sequence” Tab Zk Y Reversehighlight ( 25 basesd Probe )d Edit ” Copy Complement ” Z hQ Tools Primer Probe Test ToolY Rev Prime
12、rf Reverse Primer Y Tm=p L 58-60 d Tm= Ld Rev PrimeroYld(HY Tm, %GCqd G Y Primer (k primer/ probe design guideline)doQGYldSh ”Trim”d 1 ZJoY d%hYf Rev PrimerdQ ”Sequence” Tab Zhighlight d ” Reverse Primer” x dY Rev Primer6 z j f Primer Express Operation Guide primer/ probe set Copy & Paste 7 hQSH - Y
13、 textd( save Jf$.dp document dY File Save Asf Primer Express Operation Guide SYBR Green I Primer for Quantification Automatically Design Primer Express 3.0 File Newo “TaqMan MGB Quantification TaqMan Quantification” OK Tools 2 “Add DNA File” d 0_d2h ”Add”d Z dcfu ”Sequence” Tab4 Copy & Paste7 h if *
14、 Primer Express Software% a .dan, .txt, .ab1, .abiY , + Z Y qC(J database downloadh , ) Primer Express Operation Guide o ” Double Strand “, C6j double stranded DNA sequence ToolsjYo 1. Exclude:Y ”Exclude Selected Bases” Z - o_Y do “Exclude”dC 9 Primer Express Operation Guide 2. Junction: 8jjunction
15、”Junction” - o juctionY ( 2 bases)do “Junction”dC 9 z f 3. ”Parameters” TabQ Primer Tm= I Primers Primer Express Operation Guide Q ”Sequence” TabdTools Find Primers/Probes d C 0 G Y Primers/Probe pairs Primer Express 6Q Candidate Primers & Probe pairsdS search Q 50G d , ”Primers / Probes” Tabd !J SY
16、BR Green primer d probed% primer f ”Location” primers & probesY djY dh f ”Sequence” Tab6j ”Primers / Probes” TabYS Primers/Probe: j lJProbeYd Forward Primerd l Reverse Primerdhjf (*(j 3Y ) Primer Express Operation Guide ”Primers / Probes” Tab6 Z Primers/ProbeY d ? o G Y d?o1 K 2+ primer/ probe desig
17、n guidelinef * SYBR Green primer !5 primer dimerY * - ”Primers / Probes” TaboS primer setd hH Primer Secondary Structured Hairpin, Self Dimers Cross DimersY 1do s sY dIfGCY s sf Primer Express Operation Guide primers set dY File Save Asf ZoY primers set d Export Order InfoYdk 50HPrimers/Probes do Ex
18、port Primers/Probes ListdjG .txtdExcel f Primer Express Operation Guide Manually Design , 0Q Primer d Primer Y SH-Y Quantification Documentd( Y (k K 13+ )f 1. Forward Primer : ”Sequence” Tab Zk Primerhighlight ( 25 basesd1 )d Edit Copy (Ctrl+C) d 1h Tools Primer Probe Test Tooldo Y document type (C
19、“TaqMan MGB Quantification TaqMan Quantification”)d(Parameter ”Default”d Paste (Ctrl+V) Z h Fwd Primerd CH Tm, %GCqJIGf Forward PrimerY Tm= 58-60 d Tm= Ld Fwd PrimeroYl d( H Y Tm, %GCqd G Y Primer (k primer/ probe design guideline)doQGYldSh ”Trim”d 1 ZJoY d%hYf Primer Express Operation Guide 2. Reve
20、rse Primer : ”Sequence” Tab Zk Y Reversehighlight ( 25 basesd1 )d Edit ” Copy Complement ” Z hQ Tools Primer Probe Test ToolY Rev Primerf Reverse Primer Y Tm=p L 58-60 d Tm= Ld Rev PrimeroYld(HY Tm, %GCqd G Y Primer (k primer/ probe design guideline)doQGYldSh ”Trim”d 1 ZJoY d%hYf Primer Express Oper
21、ation Guide 3. SYBR Green primer !5 primer dimerY * - Primer Probe Test Toolo primer setdo ”Show Secondary Stucture”dC hH PrimersYZ t d Hairpin, Self Dimers Cross DimersY 1d5 dIf GCY s sd primerZ t d - 0G f Fwd Primer d Q ”Sequence” Tab Zhighlight d ” Forward Primer” x dY Fwd Primer6 z f Primer Expr
22、ess Operation Guide Rev PrimerdQ ”Sequence” Tab Zhighlight d ” Reverse Primer” x dY Rev Primer6 z j f primer/ probe set Copy & Paste 7 hQSH - Y textd( save Jf$.dp document dY File Save Asf Primer Express Operation Guide Primers & Probes for Allelic Discrimination Automatically Design Primer Express
23、3.0 File Newo “TaqMan MGB Allelic Discrimination TaqMan Allelic Discrimination” OKfD4 TaqMan MGB probe d:Q3f Tools 2 “Add DNA File” d 0_d2h ”Add”d Z dcfu ”Sequence” Tab4 Copy & Paste7 h if * Primer Express Software% a .dan, .txt, .ab1, .abiY , + Z Y qC(J database downloadh , ) Primer Express Operati
24、on Guide z SNP Z SNP highlightdEdit Annotate “SNP Target ” do SNP Y (variant)f j 528J G/A Y SNPdo Gq A Y ”R”d 2h OKdSNPC z 3Y rf Primer Express Operation Guide Tools Find Primers/Probes d C 0G Y Primers/Probes pairs Primer Express 6 Q Candidate Primers & Probes pairsdS search Q 50G d , ”Primers / Pr
25、obes” Tabf ”Location” primers & probesY djY dh f ”Sequence” Tab6j ”Primers / Probes” TabYS Primers/Probes: j lJProbe 1Yd l Probe 2Y (2H Probe Y6O $)d Forward Primerd l Reverse Primerdhjf (*(j 3Y ) Primer Express Operation Guide ”Primers / Probes” Tab6 Z Primers/ProbeY d ? o G Y d? o1 K 2+ primer/ pr
26、obe design guidelinef MGB ProbeY o du ”Parameters” Tab o ”C ” ” G ”Y* SoYf primer/ probe set dY File Save Asf ZoY primer/ probe set d Export Order InfoYdk50H Primers/Probes do Export Primers/Probes Listd jG .txt d Excel f Primer Express Operation Guide Manually Design , 0Q Primer/Probe d Primer/Prob
27、e Y SH-Y Allelic Discrimination Documentd( Y (k K 23+ )f 1. Probe for Allele 1: ”Sequence” Tabd Z SNP highlight dEdit Annotate 2 “SNP Target ” d o SNP Y (k K 24+ )f Z Probe1khighlight ( 1325 bases)d Edit Copy with Allele 1 (Ctrl+Shift+W) d Tools Primer Probe Test Tool do Y document type (C “TaqMan M
28、GB Allelic Discrimination TaqMan Allelic Discrimination”)d( Parameter ”Default”d Paste (Ctrl+V) Z h Probe1d Allele 1 variant base 3Y ( ”g”)d CH Tm, %GCqJIGf W : probe KSH Gd& CYf G Primer Express Operation Guide Tm t = (65to 67)d Probe 1oYld(HY Tm, %GCqd G Y Probe (k primer/ probe design guideline)f
29、o QGYldSh ”Trim”d 1 ZJoY dhY Probef SNP Z SNP Probe gS Y 9 Q 3L 2H base Y 9 d H basedhj Primer Express Operation Guide 2. Probe for Allele 2: ”Sequence” Tabd Z Probe2khighlight ( 1325 bases)d Edit Copy with Allele 2 (Ctrl+Shift+M) d Tools Primer Probe Test Toold Paste (Ctrl+V) Z h Probe2d Allele 2 v
30、ariant base 3Y ( ”a”)d CH Tm, %GCqJIGf Tm t = (65to 67)d Probe 1oYld(HY Tm, %GCqd G Y Probe (k primer/ probe design guideline)fo QGYldSh ”Trim”d 1 ZJoY dhY Probef!probe1q probe2 Y Tm= t 1 jf Primer Express Operation Guide 3. Forward Primer : ”Sequence” Tab Zk Primerhighlight ( 25 basesd Probe )d Edit Copy (Ctrl+C) d1h Tools Primer Probe Test Toold Paste (Ctrl+V) Z h Fwd Primerd CH Tm, %GCqJIGf Forward PrimerY Tm= 58-60 d Tm= Ld Fwd PrimeroYl d( H Y Tm, %GCqd G Y Primer (k primer/ probe design guideline)doQGYldSh ”Trim”d 1 ZJoY d%hYf
