Designing TaqMan® MGB Probe and Primer Sets for Gene
Contents
1. NOTE Because of the asymmetric placement of the minor groove binder at the 3 end complementary TaqMan MGB probes do not necessarily have the same Tm as sense probe sequences As shown in this tutorial it is necessary to test the Tm of complement TaqMan MGB probe sequences in a TaqMan MGB Probe Test Document KS Applied For Reference Onl Page 7 of 15 BS Biosystems a Designing the TaqMan MGB Probe as a complement sequence Return to the Sequence tab Again click and drag to highlight a portion of sequence approximately 20 bp long keeping the junction site towards the middle third of the highlighted region Select Copy Complement from the Edit menu Hi Primer Express 2 0 Click in the Probe 2 text box and then select Paste from the Edit menu Check the Tm of the sequence A TaqMan MGB Probe for gene expression should have a Tm of 68 70 C a a a ne __ EE TaqMan MGB Probe Test 1 o ECATGITCGATGCICGGT TPAC CPACSTC GAC AT EC If the Tm of the potential probe sequence is too high use the mouse to highlight a portion of the sequence in the TaqMan MGB Probe Test Document KS Applied For Reference Onl Page 8 of 15 BS Biosystems a The TaqMan MGB Probe Test Document will now report the Tm of only the highlighted portion of the sequence TaqMian MGB Probe Test 1 ECCATGTICGATCTCGGT When you have a sequence that has an appropriate Tm and also meets the guideli2. Software NOTE When selecting sequences for the forward and reverse primers consider the guideline for amplicon size 50 150 bases Select primers close enough in proximity to the probe to stay within this guideline First copy the final probe sequence from the TaqMan MGB Probe Test Document and paste it into a text document From the Sequence page use the mouse to click and drag over a portion of sequence upstream of the probe approximately 30 bp in length Make sure the last 5 bases of the 3 end of the sequence contain no more than 2 total G C select Copy from the Edit menu Open a Primer Test Document through the File New Menu Paste the sequence into the Forward Primer text box If the Tm is too high use your mouse to highlight a portion of a putative primer sequence in the Primer Test Document until you find a primer that meets the design guidelines as described on page 4 10 of the Primer Express v2 0 User s Manual These guidelines are also listed below 1 Avoid runs of an identical nucleotide This is especially true for guanine G where runs of four or more Gs should be avoided 2 Design primers as close as possible to the probe without overlapping the probe 3 Keep the G C content within 30 80 4 Select primers with a Tm of 58 60 C 5 The five nucleotides at the 3 end should have no more than two G and or C bases When you have a sequence that meets the guidelines select Trim from the Edit menu
3. This will delete the unhighlighted portion s of the sequence and only a primer sequence that satisfies the guidelines will remain Copy the new primer sequence Paste it in to a text document containing the probe sequence and label it as the forward primer The process can be repeated for the reverse primer this time selecting a sequence region downstream of the probe and using Copy Complement to copy the sequence into the Primer Test Document lt Applied For Reference Onl Page 13 of 15 BS Biosystems j NOTE Remember that the reverse primer will fall on the antisense DNA strand With that in mind be sure to use the Copy Complement function from the Edit menu in place of the Copy function when manually selecting a reverse primer sequence Ordering Primers and TaqMan Probes Ordering instructions for North America customers only international customers should contact their local Applied Biosystems sales office International contact information can be found at http www appliedbiosystems com about offices cfm To order Applied Biosystems reagents including primers and TaqMan probes go to the Applied Biosystems store at http store appliedbiosystems com Note you must register to be able to login and order Applied Biosystems reagents via the web Once the registration has been filled out Applied Biosystems order administration will send an e mail within 48 hours confirming your registration and you will then be able
4. select Trim from the Edit menu This will remove the unhighlighted portion s of the sequence and only a probe sequence that satisfies all of the guidelines will remain Primer Express 2 0 File Options Window Help Redo Trim Chl Cut Bi eee Van MGB Probe Test 1 Oe gt hae Copy Ctrl C Copy omplement rc lt C Ct 7 Paste Ctrl ed W Lin Clear g TGC S0 Length 18 Select All Ctrl D a0 lear All Sanotatorns p T i Pre a Find Sequence Etri HE 100 Hind Target Etri 1 Hind and E clude Etri E 150 Show Page Breaks a aATGCITTCCGATCTCGGT Show Clipboard La 200 T Preferences a Define Penalty Score TGOMGCATGT TCGATGTCGG TGECCAGCGG GACGAGCGTA GG4A4S4TGGAT 250 ACGTCGTACA AGETACAGCC ACOGGTCGCC CTGCTCGCAT CCTTTACCTA NSS Applied For Reference Onl Page 6 of 15 BS Biosystems d j If all of the TagMan MGB probe design guidelines cannot be satisfied ex if it is not possible to select a probe without a guanine residue at the 5 end you will need to design a probe on the complement antisense strand If it is not necessary to design the TagqMan MGB probe on the complement strand go to the section entitled Adding a Probe Annotation amp TaqMan MGB Probe Test 1 Note This TagMan MGB probe does satisfy all of the recommended guidelines However in Some cases designing a complement probe may be necessary to satisfy all of the TagMan MGB probe design guidelines
5. electronic orders The Order document is a text file that enables the editing of the sequence information for ordering If the TagMan MGB probe was designed as a complement sequence the TaqMan MGB probe must be re entered in as the complement sequence To edit the Order document copy the final probe sequence complement from the TaqMan MGB Probe Test Document Return to the Order document and delete the current probe sequence Select Paste from the Edit menu E DROGHEPSA3 gb TagMan MGB Pr Order Please synthesize the following oligos for ne and bill my account Hame DROUDHEFSAI ghb LILF Sequence CAGTAAGTGTAATCCCAAGTGATATCG Note If selecting the Name DROGNDPSAJ gb 264R comp lement probe JENTE CATTGAAACACTGAATCCATTTECT sequence the and the Taqtan probe Neame DROGHEPSAS gb 206TSequence i ACCGACATCGAACAT SG wooli sequence in the Order Thanks very much document must be prazinse deleted and the complement sequence must be re entered P f B The sequences within the order document can now be copied and pasted into electronic orders lt Applied For Reference Onl Page 12 of 15 BS Biosystems d There may be times when the Primer Express Software is unable to find primer sets in conjunction with your manually designed probe If no acceptable primer sets were found you will need to perform a manual design within the Primer Express Software Manually designing primers through the Primer Express
6. the middle third of the highlighted sequence From the Edit menu select Copy Under the File New menu select TaqMan MGB Probe Test Document Primer Express 2 0 File lenkai MEL le BE Te ee climerit Sema Ma EEDI tiie TTCACCTOIT TCATITGAAA GTCATTCACA 7 Cad TATTTTAGTT TCTATTCCAT TCTAATGATA 1 CTATAGCGTT ATAAAATC AA AGATAAGGTA AGATTACTAT i a aemm Click in the Probe 1 text box and then select Paste from the Edit menu TaqMan MGB Probe Test 1 Ox BOCATOCITCCATCICGGCIOG AR oP pplied For Reference Only Page 5 of 15 Biosystems Check the melting temperature Tm of the sequence TaqMan MGB Probes for gene expression should have a Primer Express estimated Tm of 68 70 C TagMan MGB probes for gene expression should also be designed following the guidelines listed below 1 Keep G C content in the 30 80 range 2 Avoid runs of an identical nucleotide This is especially true for guanine where runs of four or more Gs should be avoided 3 Do not put Gs on the 5 end 4 Make TaqMan MGB Probes as short as possible without being shorter than 13 nucleotides If the Tm of the potential probe sequence is too high use the mouse to highlight a portion of the sequence in the TaqMan MGB Probe Test Document The TaqMan MGB Probe Test Document will now report the Tm of the highlighted portion of the sequence When you have a sequence that has an appropriate Tm and also meets the guidelines above
7. to place an order To order primers and TaqMan probes click on ABI PRISM Primers Probes in the catalog column left hand column Then click on TaqMan Primers amp Probes in the catalog column Scroll down to locate the items that will be purchased For the products that are to be ordered check the boxes located on the left hand side of the product names Once all of the products have been selected click on the Add to Shopping Basket button The products will now be itemized in the shopping basket It is important to enter the sequences of the primers and or probes Click the not customized button next to the product name for each custom primer and TaqMan probe Follow the instructions to enter in the sequence of the primer or TagMan probe Repeat this process for all custom primers and TaqMan probes To process the order click on the Process Order button and fill out the requested information to complete the order Ordering questions for primers and TaqMan probes can be directed to the Applied Biosystems Custom Oligo ordering group at 800 327 3002 Follow the touch tone menu to speak with an Order Administration representative regarding primers and TaqMan probes Ordering questions for SDS Real Time PCR reagents and consumables can be directed to the Applied Biosystems Order Administration group at 800 327 3002 Follow the touch tone menu to speak with an Order Administration representative regarding r
8. window Help DROGNBPSA3 9b TaqMan MGB Pi Primers Map Recipe Results Revers Primer Amplicon m GG Primer Length Tm 6C Ta Penalty Click on the ai 260 CATT OMACACTO ATC CATTTICCT Ez T 4 5 mA hoe 40 CATGAAaC ACTG E 77 EE B 433 0 line containing Saroee TOAST CCATTICC 134 77 43 si 431 0 i 56 wy TATTIOAAAL ACTOAATCCATITCCTA 135 77 4 5 499 0 the Primer and EE 38 CATTGAAACACTGAATCCATTTEC iM 77 42 5 470 T a0 CATTGAAADACTGAATCCATTITCCT E 77 42 57 438 0 probe set of 59 30 CATTIGAAACACTGAATCCATTITCCT 137 77 43 FA 449 0 z 59 35 CATTIGAAACACTOAATCCATITCCTA 137 77 42 Bei 450 0 Interest 5a 38 CATTOAAALACTOAATCCATTICL 137 77 4 S 440 0 a 30 CATTGAAACACTGAATCCATTTECT E 77 a3 Bj 445 0 is ct4 CAaITTGS44C 46 C TGS4ICCATTICL FE rf ay baa 4 0 59 35 CATIGAAACACTOAATCCATITCCTA E 77 43 rN 456 0 re kone Order Save List Display Atotal of 7 primer pairs Toure ck any entry inthe ict to gelect R Detalls in Primer Data wiriou Order Button NS Applied For Reference Onl Page 11 of 15 AS Biosystems d i Click on the Order button at the bottom of the Primers tab DROGNBPSA3 gb TaqMan MGB Pr Order Name DROGNBPSAS gb l31F Sequence CAGTAAGTGTAATCCCAAGTGATATCG Name DROGNEPSAS gb 264F sequence CATTGASACACTGAATCCATTTICCT and the TaqMan probe Name DROGHBPSAS gh 206TSsequence CATGTTCGATGTCGGT Thanks very much This order form does not actually place
9. Designing TaqMan MGB Probe and Primer Sets for Gene Expression Using Primer Express Software Version 2 0 Overview This tutorial details how a TaqMan MGB Probe can be designed over a specific region of a template sequence such as an exon exon junction intron splice site Genomic DNA is often co extracted with RNA and can therefore serve as a template in downstream processes such as PCR Designing a TaqMan MGB Probe over an exon exon junction should enable the exclusion of genomic DNA as a template in a real time PCR reaction This tutorial assumes basic working knowledge of the Primer Express v2 0 software If you are unfamiliar with the software please first review the following documents e Primer Express Software v2 0 User s Manual document part 4329500 e Primer Express Software v2 0 Applications Tutorials document part 4329501 Note The documents above can be found electronically on your hard drive in Program Files Applied Biosystems Primer Express Starting the Design and Entering the Sequence For gene expression assays it is best to enter a cDNA sequence or a mRNA sequence into the Primer Express Software If entering a mRNA sequence and the sequence contains Uracils U one must first convert the Us to Thymidines T in a word processing document To replace the Us with Ts Copy and Paste the sequence into a word processing document such as Microsoft Word Go to the Edit menu and select Replace In th
10. TEIT TCATIOGAAA GTCATTCAGE TTAGGSTICA GATATCOCAR TATTTIACTO TCTATECCAT TOTAATOCATE TOCATICOTG CTATMROCOTT ATAAAATC RR AQATAWHITA AGATTACTAT ACOTAROCAC ATGT TCGATETOSG TGSCCAGCOG GACGAGCETA GGAAATOGAT Double Stranded ACETOCTACA BAL TACLABOC ACCEETCOLE CMATCCCAT COTTTACCTA CAGTTTTC AATOATETAA CTECTATCAT ATTCOTAACT GCGTCCTCAA DNA check box ACTCACAAAD TTACTACATT GACDATAOTA TRAGCATTOR COCADGROTT GTTATAACAT GGTTTTGOS GAAGATCOCA DOCAGAACDS ACTTOGACAA CAATATTIOTA TCCAAAACEOD CTTCTAGGGT GSGTCTIGSC TGAAGCICTI AOAAACCTAA ACAAQTTCTC ATAAACCTTIO TTOTCCACIT OOCTOCTTAA ACCBORFTAG KATETTA CTTTRATTOO TCAACAADAE AGTCTACCOA TCECACGATT TOTATTATAC TATTTOTAAA TRACCAACAT TICTTAGCAG ADCCTOCTAA BGATAATATG ATAARRATTT ATOCOTICTS AACAATOOTC ADAREATTAA GATOR ACTAARTTOT DSGANTATIO CTCCRRGTIT TTAATT TORACCTIOG TCATITAACA GOCTTATAAE GAGGCTCAAA AACRRATACC RRACCCCRR TAADTAAAAT BROTATAACOS ATACTAGACG TIGTIYTATOO TITUCOCTHC ATTCATTTTA TCATATTOGS TATCATCTCC KS Applied For Reference Onl Page 3 of 15 BS Biosystems a Use the Line tool to mark the junction site in the sequence This only provides a visual aid and does not direct the software to design the primer probe set over the site TGCAGCATGT TCGATGTCGG TGGCCAGCGG GACGAGCGTA GGAAATGGAT 220 ACGTCGTACA AGCTACAGCC ACCGGTCGCE CTGCTCGCAT CCTTTACCTA Annotated Junction Site Click the Params tab Click the Defaults button Ideally you will not have to alter the parameters as the default para
11. cture Hide Statue Bar Copy Page To Window lt Applied For Reference Onl Page 10 of 15 AS Biosystems d If the software finds acceptable primers click the Primers tab If the software cannot find acceptable primers skip the following and proceed directly to the next section entitled Manually designing primers through the Primer Express Software Primer Express 2 0 File Edit Options Window Help A DROGNEPSA3 gb TaqMan MGB Pr Sequence File Name DROGNEPSA2 gb mpat GHA File Length 1380bp Selection FAO to RAD AAGCTITCCTO GACCGAGTGA GCACAATCAA GAATCCAAAC TACACCCCTA TTCGAAGCGAC CTGGCTCACT CGTGTTAGTT CTTAGGTTITG ATGTGGGGAT ATGAGCAGGA TATTCTTCGG TGCCGTGTTT TGACTTCTGG AATATTTGAA TACTCGTCCT ATAAGAAGCC ACGGCACAAA ACTGAAGACC TTATSS amp acTT ACAAGATTTC SAGTGGACAA AGTAAACTTT CAGTAAGTGT AATCCCAAGT TGTTCT amp 44G6 TTCACCTGTIT TCATITCAAA GTCATTCACAaA TTAGGGTTCA Foa TATTTTAGTT TCTAaATTCCaT TCTAATGATA TGCATTCOTG CTATAGCGTT ATAASATCAA AGATAAGGTA AGATTACTAT ACGTAAGCALC TGCAGeatgt tegatategg tGGCCAGCGG GACGAGCGTA ee ALATGGAT ACGTCqtaca aggtacagec aCCGGTCGCC CTGCTCGCAT AGTGTITC AaTGATGTAAa CTGCTATCAT ATTCGTA amp ACT GCGTGCTCAA LGTCACASAG TTACTACATT GACGATAGTA TAAGCATTCGCA CGCACGAGTT Select a primer pair from the list that will produce the shortest amplicon while satisfying all design guidelines Click on the line containing the chosen primer and probe set Fim Primer Express 2 0 Fle Edt Options
12. e Find what field type the letter U In the Replace with field type the letter T Click on the Replace All button The sequence which now contains Ts instead of Us can be copied and pasted into the Primer Express Software Sequence tab Unless one is designing from a single exon gene it is not recommended to use genomic DNA sequences for the design of gene expression primer and probe sets lt Applied For Reference Onl Page 1 of 15 AS Biosystems j For a gene expression assay select the TaqMan MGB Probe and Primer Design document from the File New menu Primer Express 2 0 er eb Epon Print Setup TagMan MGB Probe amp Primer Design mjeri Ma S litt Ciher Send Mei Ciis lt gt Applied For Reference Onl Page 2 of 15 BS Biosystems j Enter the sequence either by going to File Import using the Import DNA File button in the Sequence Tab or by copying and pasting the sequence through the Edit menu Primer Express 2 0 File gave i if at Import Ga ph Export sena Mail Etrit Next if not checked Click in the Double Stranded and Limit 3 G C boxes Limit 3 G C AKBCTTCCTG GACCGAGTGA GCACRATCAA GRATCCARAC TACADCCOTY 7 check box DERHAL TTAPLT CAT COTSTTAGTT CITACE ITE ATOTOoS ATCA ACCA TATTCTTO2e TOCCETOTTT TEACTICTOS AATATIOGAA TACTOGTCCT ATAAGAABOC ACODCACAAA ACTOAAGACC TTATAAACTT ACARBGATTIC BMGTGGATEA AGTRALC TTT CAGTAAGTST AATCOCLAGT AAAG TICACC
13. eagents and consumables Technical questions about Applied Biosystems SDS Real Time PCR reagents and consumables including primers and TaqMan probes can be directed to the Applied Biosystems PCR amp SDS Technical Support group at 800 762 4001 lt Applied For Reference Onl Page 14 of 15 BS Biosystems d 2002 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures The PCR process and 5 nuclease process are covered by patents owned by Roche Molecular Systems Inc and F Hoffmann La Roche Ltd Applied Biosystems ABI PRISM and Primer Express are registered trademarks and AB Design Applera and FAM are trademarks of Applera Corporation or its subsidiaries in the US and certain other countries TaqMan is a registered trademark of Roche Molecular Systems Inc Microsoft is a registered trademark of Microsoft Inc lt Applied For Reference Onl Page 15 of 15 BS Biosystems j
14. meters follow the TaqMan Probe and Primer design guidelines as established by Applied Biosystems For a list of the design guidelines please refer to page 4 10 of the Primer Express Software v2 0 User s Manual One additional guideline specific to TaqMan MGB probes is that the probe should be as short as possible without being shorter than 13 nucleotides TaqMan MGB Probe 1 Ml Sequence Params RxnCond Primers Map Recipe Results Primer Tm Requirements win Tm l Sg hax Tm BO Optimal Tm 59 hiaximal Tm difference F Primer GC Content Requirements Win GC 30 Max BGC B0 F GCclampof O residues Primer Length Requirements h n length s hitax length g Optimal length 20 Amplicon Requirements hin Tm a hax Tm a5 hin length 50 htx length 150 TagMan MGB Probe Criteria Taghtan MGB Probe Tm must be 10 0 greater than PCR Primer Tm M Tagtan MGB Probe should not begin with G More Params Defaults Factor Defaults Return to the sequence page by clicking on the Sequence Tab You are now ready to manually design the TaqMan MGB probe lt Applied For Reference Onl Page 4 of 15 BS Biosystems d j Designing the TaqMan MGB Probe Make sure that the junction site is still labeled If not label the junction site using the Line tool Using the mouse click and drag to highlight a portion of sequence approximately 20 bp long keeping the junction site towards
15. nes on page 6 select Trim from the Edit menu This will remove the unhighlighted portion s of the sequence and only a probe sequence that satisfies all of the guidelines will remain TaqMan MGB Probe Test 1 BELATGITCGATCTC GGT EC CACATC PARC AT Y Applied For Reference Onl Page 9 of 15 BS Biosystems a Adding a Probe Annotation Return to the Primer Express Software Sequence page Select the Probe tool and then highlight the portion of sequence matching the TaqMan MGB probe ACAADATT I AAGTIGACAA AUTAAACTTT CAGTAAGTOT AATCCCAAGT Lo TGTTCTAAAG TICACCTGTT TCATTTGAAA GICATTCACA TTAGGGTTCA GATATCGCAA TATTITTAGTT TCTATICCAT TCTAATGATA TGCATTCGIG 200 CTATAGCGTT ATAAAATCAA AGATAAGGTA AGATTACTAT ACGTAAGCAC S equence TGCAGCatyt tegatgtegg CGCCAGCGG GACGAGCGTA GGAAATGGAT 250 ACGTCgtaca auctacagc accGeTCGLe CTGCTCGCAT COTTTACCTA annotated with BS YTCAGTCTTIC AATGATGTAA CTGCTATCAT ATTCGTAACT GCGTGCTCAA 300 probe tool AGTCACAAALD TIACTACATT GACGATADTA TAAGCATTGA COCACGAGTT GITATAACAT GGOTTTTGCGG GAAGATCCCA CCCAGAACCG ACTTCGOAGAA 350 CAATATTGTA CCAAAACGCE CTTICTAGGGT GGGTCTIGGC TRAAGCTCIT Designing the Primers Ensure that the Limit 3 G C checkbox is still checked Select Find Primers Probes Now from the Options menu Som Primer Express 2 0 File Edit S800 windon Help Turn AutoFind ON Find Primers Frobes Now Hide Annotation Tools Hide Primer Data Show Interim Results Show Primer Secondary Stru
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