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1、Primer Premier6.0ManualPREMIER Biosoft International3786 Corina Way,Palo Alto, CA 94303-4504Tel: (650) 856-2703FAX: (650) 618-1773E-mail: Copyright 2009 by PREMIER Biosoft International. All Rights Reserved.Information in this manual may change without notice and does not represent a commitment on t
2、he part of PREMIER Biosoft International.The software described in this manual is provided by PREMIER Biosoft International under a License Agreement. The software may be used only in accordance with the terms of the agreement.PREMIER Biosoft International (Premier) claims copyright to this program
3、and documentation as an unpublished work. Claim of copyright does not imply waiver of Premiers other rights.This program and documentation are confidential and the property of Premier. Use, examination, reproduction, copying, decompilation, transfer, and/or disclosure to others are strictly prohibit
4、ed except by express written agreement with Premier.For more information on Primer Premier and product updates, visit the PREMIER Biosoft web site at http:/www.PremierBiiPrimer Premier 6.0Introduction to Primer Premier1WORKING ENVIRONMENT3Menus and Toolbar3Main Window9The main window displays the Se
5、quence Information Tab, Search Status Tab, Sequence Viewand the Search Results Tab. Sequence Information Tab9Primer Search Parameters11BLAST Search Parameters14Template Structure Search19View Cross Homology20View Secondary Structures22View Template Secondary Structures23Multiplex Results24TUTORIAL25
6、Create and Manage a Project25Create New Project25Open Project25Close Project25Save Project As25Save Sequence As26Open Sequence27Sequences can be opened directly from Entrez, dbSNP or from files saved on your local drive.Open Sequence from Entrez27Open Sequence From dbSNP27Load Multiple SNP29Add SNP3
7、0Select/Unselect SNP30Search Sequence31Avoid Cross Homology32Interpreting Cross Homology Results36Interpreting Human BLAST Results37Interpreting Microbial BLAST Results38Avoid Template Structures39PCR Primers41Primer Search Results43Export Results45Evaluate predesigned primer46Multiplex Primers48Gen
8、erate Report49Print Preview50PREFERENCES51Reaction Condition51APPLICATION PREFERENCES52iPrimer Premier 6.0TECHNICAL INFORMATION53Internet Setting53Primer Premier Automatic Upgrade54Standalone WWW BLAST for Linux55Install RedHat Linux Server56Download BLAST Binaries59Setup Standalone WWW BLAST Server
9、 for Linux60Format Databases62Configuration Files64System Requirements65Troubleshooting66Standalone WWW BLAST for Mac69Install Standalone WWW BLAST Server for Mac70Setup Standalone WWW BLAST Server for Mac71Download Databases72Format Databases for Mac73Configuration Files for Mac75System Requirement
10、s for Mac76Troubleshooting for Mac77Algorithm and Formulae79Formula for Melting Temperature Calculation79Formula for Calculating TaOpt81Formula for Free Energy82Formula for Rating83System Specifications85Suggestion86Software Limits87Terms of Use88REFERENCES89iiPrimer Premier 6.0 Introduction to Prim
11、er PremierIntroduction to Primer PremierPrimer Premier is the most comprehensive tool for designing and analyzing PCR primers. Primer Premier 6 designs primers for standard PCR. It automatically interprets the BLAST search results and utilizes a proprietary algorithm to check for possible secondary
12、structures. Homologies and structures are avoided for designing highly specific and efficient primers. Search algorithm finds optimal PCR, multiplex and SNP genotyping primers with the most accurate melting temperature using the nearest neighbor algorithm. Primers are screened for secondary structur
13、es, dimers, hairpins, homologies and physical properties before reporting the best ones for your sequence, in a ranked orderPrimer Premier provides the following major functionalities:Primer designOptimal primers designs primers free of dimers, repeats and runs.Multiplexing Pools of primers are chec
14、ked for cross homologies to reduce primer dimer.Allele Discrimination designs primers for detection of both wild and mutant alleles.SNP amplification designs SNP flanking primers to amplify SNPs.Multiplex primers checks primers for cross reactivity preventing competition in multiplex reactions.Evalu
15、ate pre-designed primers allows the use of previously designed or published primers of standard PCR assays. Designs a compatible primer given an antisense or sense primer.Avoid cross homology ensures specificity by automatically avoiding homologies found using BLAST.Primer and amplicon BLAST search
16、BLAST searches primers and amplicons to verify specificity of the design.BLAST search BLAST searches the entire sequence, designed primers and amplicon to visualize specificity.BLAST database searches local custom databases using StandAlone or Desktop BLAST or connects directly to the NCBI server fo
17、r public databases.Repeat and low complexity regions optimizes BLAST search parameters to detect repeats and low complexity regions while searching the genomic databases available at NCBI.BLAST result view provides result view for BLAST searched on the sequence, primer-pair and amplicon.Algorithm ca
18、lculates highly accurate Tm using nearest neighbor thermodynamic algorithm. Rating ranks primers as per their priming efficiency and screens them for their thermodynamic properties as well as secondary structures.Database ManagementProjects creates multiple projects. Data of multiple experiments can
19、 be easily managed by creating separate projects for each experiment.Application database maintains a local database for sequence information and search results.1Primer Premier 6.0Web IntegrationWeb Integration batches of sequence can be quickly retrieved from Entrez using accession numbers and from
20、 dbSNP using assay Ids.BLAST Search BLAST searches sequences against local custom databases using the StandAlone BLAST service and Desktop BLAST from NCBI.Template Secondary structure search uses an innovative proprietary algorithm to detect and display the possible template structures at folding te
21、mperatures. These structures are avoided during primer design.Input/OutputOnline sequence retrieval automatically connects to Entrez or dbSNP and retrieves multiple sequences.File format supports GenBank, FASTA and dbSNP formats. GenBank and FASTA formated files can contain multiple sequences.SNP Lo
22、ading - easily adds SNP(s) at a time on a single sequence or load thousand of SNPs using standard GenBank variation files.View output in spreadsheet results can be viewed and manipulated in any spreadsheet program like MS Excel and lotus 123.Flexible output format tab delimited format of the output
23、file can be easily loaded in to any database.2Primer Premier 6.0 Working EnvironmentWorking EnvironmentMenus and ToolbarThe Primer Premier toolbar is displayed at the top of the main window under the menu bar. It provides shortcut buttons for the following menu commands. The toolbar options can also
24、 be accessed from pop-up menu at right click (For Macintosh, CTRL+click) over the Sequence Information tab and Search Status tab.MenusThe main menu bar is at the top of the main windowMenuToolbarHot KeysHotkeysDescription(For(For MAC)windows)Primer Premier 6.0Program versionand PREMIERBiosoft contac
25、tinformationAbout Primer PremierQuit Primer PremierFile MenuAlt+FNewNew ProjectCtrl+N+NCreate a newproject to managethe data of everynew designproject.New SequenceCtrl+E+EAdd a textsequence from anyother applicationby cutting andpasting thesequence bases orby manually typingthe sequence inthe Sequen
26、cewindow.OpenProjectCtrl+O+OOpen to work onan existing project.Sequence:From FileCtrl+L+LRetrieve oneSingle or multiple3Primer Premier 6.0 Working Environmentmore sequencesfrom a local file.(must be in FASTAor GenBank format)From EntrezCtrl+G+GRetrieve one ormore sequencesfrom Entrez.Program interpr
27、etsthe SNPinformation in theheader anddisplays itFrom dbSNPCtrl+S+SRetrieve one ormore sequencesfrom dbSNPSave Project AsSave the existingproject withdifferent file nameat specifiedlocationSave Sequence AsSave the existingsequence withdifferent namewithin the projectClose ProjectExportCloseandsaveth
28、e current project.Exports the primerand probe searchresults in a tabdelimited spreadsheet format filetype(.xls,.csv).Export Primer ResultsCtrl+Alt+EAlt+ELaunch ExportResults windowfor primer searchresult export.Export SequenceCtrl+Shift+S+Shift+SLaunch ExportSequence windowfor exportingsequence with
29、/without oligos.ReportExport the designresults in a reportformat that can be4Primer Premier 6.0 Working Environmentprinted.Generate ReportThe user mayselect the data thatneeds to beincluded in thereport by checkingor un-checking theboxes of thecorrespondingfields.Open reportCtrl+Alt+N+ Alt + NOpen t
30、he reportfrom local file thatcan be printed.ExitEdit MenuAlt+ECopySense PrimerCtrl+Shift+E+Shift+EAntisense PrimerCtrl+Shift+A+Shift+ASelect AllCtrl+A+AFindCtrl+F+FDelete SequenceDeleteDeleteView MenuAlt+VSequence DetailsCtrl+Alt+Q+Alt+QBLAST DetailsComplete SequenceCtrl+Shift+S+Shift+SSaves active
31、working project and terminates program.Copy the Sense Primer of the selected sequence to the clipboard.Copy the Anti-sense Primer of the selected sequence to the clipboard.Select all sequences in the Sequence Information tab.Search Sequences.Delete selected sequences in the Sequence Information tab.
32、View selected sequence source file.View BLAST results information for the selected sequence.5Primer Premier 6.0 Working EnvironmentPrimer PairCtrl+Shift+P+Shift+PView BLASTresults informationfor the Primer Pair.AmpliconCtrl+Shift+C+Shift+CView BLASTresults informationfor the Amplicon.Template Struct
33、ure DetailsCtrl+Alt+T+Alt+TView Secondarystructureinformation for theselectedsequence.Multiplex PrimersCtrl+Shift+I+Shift+ICross HomologyCtrl+Alt+H+Alt+HAllPrimerAll StructuresPrimerSequence ViewCtrl+Alt+Z+Alt+ZBase Per BlockLaunch Multiplex Primers result window.(More than one sequences should be s
34、elected)View Cross-Homology resultsdata interpretedform the BLASTresults for theselectedsequence.View All primers for the selected sequence ordered by descending rating.View graphicaldisplays of primersecondarystructures for theselectedsequence.Show/Hide Sequence View.Shows either 3/10 bases per block in the sequence.Sequence StrandShows eitherSingle/Doublestrand in thesequence.Tool barShow/Hide Toolbar.6Primer Premier 6.0 Working EnvironmentStatus barShow/Hide Statu