PrimerBLAST 操作说明.ppt

PrimerBLAST PrimerBLAST 操作说明操作说明Two Design StrategiesUse Accession Numbers or Proprietary Genes to create gene-specific primers for amplicons 105-350 nucleotides in lengthThe shotgun approach is an efficient method to use when little or no genome information is available for the organism of interest. •Little or no homology, pseudogene or transcript variant information•No SNP information•No intron/exon boundariesThe strategic approach is recommended when a model organism is used and/or there is sufficient genomic data available in NCBI for Primer-BLAST to scan primers for:•Homology and/or transcript variants•SNP•Intron/exon boundaries are known12Accession NumbersStrategic ApproachNCBI Primer-BLAST designPre-design considerationsOrder primers with universal tags and evaluate primers in vitro•Pre-design considerations–Choosing the gene and accession number•Use only mRNA accession numbers–Amplicon length and space between peaks•Design using NCBI Primer-BLAST–Amplicon lengths–Design primers to detect all transcript variants (if not specified) or a unique transcript variant (if specified)–Design primers to avoid amplifying transcribed pseudogenes–Design intron-spanning primers when possible–Exclude SNPs from primersStrategic ApproachAdvanced Primer Design WorkflowAdvanced Primer Design WorkflowObtain mRNA accession numbers or use gene sequenceCheck gene for transcript variants, pseudogenes and intron information: Entrez Gene(See Specific Design Considerations)Design primers using Primer BLASTEnter primer name, fragment size, gene name into multiplex TDF file templateDetermine the desired length of amplicon (without universal tags)1. Choosing an Accession Number•A single gene can be represented in the NCBI database by multiple accession numbers–mRNA»Always use reference sequence (RefSeq) (NM_XXXXXX) from the Database»Use caution with other mRNA accession numbers - partial sequences- mutations- ESTs•Do not use an accession number for genomic sequences2. Ensure the following for each accession number that is chosen:•Correct gene is chosen–Multiple names and aliases –Different genes can have similar names –Species of interest –If non-RefSeq accession number is used Verify that the sequence contains only the letters A, T, G, C or NPre-Design ConsiderationsAmplicon Length•Design amplicons such that each fragment is no less than 5 nucleotides apart from its nearest neighbor.–This allows for variation in migration to meet the minimum peak separation distance of 3 nucleotides.•Design amplicons between 105 – 350 nt (without universal tags)–142 – 387 nt with universal tags–Start with small fragment size and work toward larger fragment sizeNote: For FFPE samples, design amplicons between 105-160 nt (without universal tags), the recommended total number of fragments in a panel is twenty or less.Pre-Design Considerations-Continue• Gene Information• Intron and Exon Information• Transcript Variants Information• Pseudogenes InformationPre-Design Considerations-Continue1. Choose the GeneGene database2. Enter gene name or accession number3. Click SearchNCBI web or directly go to http://www.ncbi.nlm.nih.gov/gene/Step1: Getting Gene InformationGet the alias(es) and a summaryExample: NM_01825View the transcripts and reference sequencesELP2 contains a single transcript with 22 exonsEach green bar represent an exon.ELP2 located at the Chromosome 18Step 2: Checking for homology and pseudogenesPerform a species-specific queryBlast human genome if it is a human geneGo to http://blast.ncbi.nlm.nih.gov/Blast.cgiEnter the accession # hereSelect “reference only” databaseClick “Begin Search”Click “View Report”Click View Report to view gene information Only one hit, there’s no high homologous region or pseudogenes.Click on the link to go to the map view and intron-exon information.Report view pageBlack hash marks indicate exon-exon boundariesNM_018255Go to http://blast.ncbi.nlm.nih.gov/Blast.cgiScroll down to find the Specialized BLAST, click Primer BLASTStep3: Design Gene-Specific Primers NCBI Primer-BLAST•This program will accomplish the following when the parameters are set properly:–Check for specificity both within a species and between species (must add species)–Include or exclude transcript variants –Exclude SNPs from the primer binding sites–Create intron-spanning primers and/or exon-junction primers–Allows for design to specific regions within the gene or using a pre-designed primer sequences–Avoids low complexity primer binding regions–Note: Generally the more restrictions that are placed on the primer selection program, the fewer primers binding sites will be available •This program does NOT:–Check for repeat sequences within the amplicon which may lead to stutter–Check for pseudogenesDesign Gene-Specific Primers with NCBI Primer-BLASTPrimer-BLAST3.(optional) Specify specific nucleotide regions for the forward and reverse primers2. Enter pre-designed primers (if any, optional)1. Enter accession number or FASTA sequence (mandatory)Primer-BLAST continued4. Specify product size (mandatory) range = 105 – 350 nt for XP-PCR6. Use default Tm settings1051055. Specify the # of primer sets to return (optional)8. Verify specificity by checking the box and entering the species (recommended)9. Include splice variants by checking this box (optional). Try to exclude splice variant by leaving it blank.7. Check this box to design intron spanning primers)Recommended 500 as MinClick Advanced parameter for more options12. Change Default Salt correction formula and Thermodynamic Parameters as indicated belowSalt = Schinderkraut & Lifso 1965 Tm = Breslauer et al. 198613. Click “Get Primers to obtainresults11. Exclude SNPs from the primer binding site.ResultsPrimer Design continue…Add the universe taq sequence to each of the gene specific primer sequence as the final primer sequenceExample:Forward Sequence with the Universal Taq SequenceAGGTGACACTATAGAATATGATCGGGTCTTCCTTCATCReverse Sequence with Universal Taq SequenceGTACGACTCACTATAGGGAGCCATTAAGGCCCTTCTTTCThe designed fragment size is the gene specific size plus 37bp of the universal taq, eg, the designed size is 105bp, the expected product size is 142bp.Order primers with Universal Tags, except KanR (in buffers)1. Keep the all header columns information unchanged.2. Change a new multiplex name (cell A2) when the multiplex information was modified.3. Primer name, GeXP product size, Gene Name and Accession number information are essential.4. Enter same information for both Gene Name and Accession number columns. 5. Use the actual CEQ fragment size in the product size with universal taq column6 Row 3 has to be empty7. Keep at the last line of primer name.Create a Multiplex from a TDF file templateWhat is the TDF File ?The TDF file contains the multiplex information and that links the X-Profiler Analysis with the imported GeXP csv data.A multiplex can be uploaded into eXpress Profiler from administrator account with a valid* TDF file.•Unique Multiplex Name (cell A2 in Excel spreadsheet)•Row 3 has to be empty•Keep all the column header unchanged.•Mandatory fields to be filled: Primer Name, Gene Name, Accession Number, Product Size w/ Universals (use GeXP final product size easier for binning!!).•Enter same information in the Gene Name and Accession Number (Can be gene name or accession number).•Primer name do not include ()(), otherwise the quotation marks will created when you modified the TDF, the TDF will fail to be imported.Specific Primer Design Considerations•Targeted specific transcription variants•Excluded pseudogenesExample: Targeted Primer Design for BIRC5http://www.ncbi.nlm.nih.gov/gene/Target the 5’ exon1-2 to capture three variantsGo to Nucleotide home http://www.ncbi.nlm.nih.gov/nuccore, enter accession #BRC5 gene has three transcript variantsClick GraphicsGo to NCBI Nucelotide home http://www.ncbi.nlm.nih.gov/nuccore, enter accession #Graphic to view exon information Example: NM_001012271, five exonsRight click the exon bar to view exon positionsHas to reduce the intronsize in order to designprimer between exon1-2.Primer Designed: Specific the forward primer and reverse positionsPrimer Designed: Selected “Allow to amplify splice variantsResultsPrimers targeted three transcript variantsDesign primers to regions unique to the real mRNA.Design Primers excluding the PseudogenesWhy design primers spanning an intron?•To avoid genomic DNA interference from–Samples resistant to DNase–Samples too small to be treated with DNase–Single cell analysis•To minimize or eliminate RT minus control –Saving for reagents and time Note: This step is optional or sometimes impossible step for certain multiplex panelOptional when DNase treatment will be performed routinelyImpossible when gene/variant specificity is only in the 3’UTR (no introns).Plex B RT minus controlEach primer pair was designed on different exons to avoid interference from genomic DNA contamination.No false positivePlex A RT minus controlMultiple false positives / false signalPrimers were designed without spanning intron. Multiple false positives in RTminus although samples were DNase treated.Other Considerations•Designing primers for gene family members•Designing primers for splice variants•Choosing quality reference (housekeeping) genes•XP-PCR Primer SpecificationsDesign gene-specific primers for gene family members•When gene family members are involved, proprietary sequences should be created using non-homologous sequences.•If a block of non-homologous sequence is not available, individual primer sets should be designed prior to the multiplex design. The primer should have its 3’ end land in non-homologous region. Design gene-specific primers for gene family members, when using proprietary sequence is not an option•Perform alignment•Pick a reverse primer that has its 3’ end land on non-homolog region•Paste the primer sequence into right primer window•Pick a forward primer that has its 3’ end land on non-homologous region•Paste the primer sequence into left primer window•Click Execute•This task can also be performed via Primer-BLAST athttp://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgiExample: design gene-specific primer regions for CYP6Z1Alignment of CYP6Z family Primer Design for Splice VariantsmRNAOne set of primers target common exons and amplify One set of primers target common exons and amplify multiple splice variantsmultiple splice variantsThis will only work if exon 2 is small enough (< 200 nt)1234v1134v21234120 nt300 ntAmpliconCommon reverse primer and specific forward primers Common reverse primer and specific forward primers amplify multiple transcript variantsamplify multiple transcript variants1234Now it doesn’t matter how big exon 2 is.Design an exon 1-exon 3 junction forward primer for v2120 ntnt1251234v1134v2mRNAAmpliconSpecific reverse and forward primers amplify a single Specific reverse and forward primers amplify a single transcript varianttranscript variant4123Design an exon 2-exon 3 junction reverse primer for v11234v1134v2100 ntDoes not amplifymRNAAmpliconChoosing Quality Reference Genes•Equivalent expression of each reference gene over all samples (tissues, treatments, time points) examined–GeXP Human ReferencePlex•No psuedogenes present or target a unique region in the reference gene•Use multiple (3+) reference genes for normalization–Add more than and then choose which ones to use for studyHousekeeping (Reference) Genes Primers availableTBP (NM_003194)170bp, 212bpPSCM4(NM_153001)143bp, 271bpHRT1(NM_000194)234bpCCNG1(NM_004060)278bpGUB2 (NM_000181)198bpRPLP0(NM_053275)115bp, 127bpGAPDH(NM_002046)248bpMousemTBP(NM_013684)151bpmGUB2(NM_010368)238bpmCCNG1(NM_009831)281bpmGAPDH(NM_008084)349bpHumanGAPDH has many pseudogenesIt is impossible to design primers around all the pseudogenes.XP-PCR Primer SpecificationsFeatureminoptimal maxunitTm*57 6063 oC Length17 2023 nt Amplicon Size105 -350nt*The default values for "Table of thermodynamic parameters" and "Salt correction formula" (under advanced parameters) have been changed in NCBI Blast to values recommended in primer3 program. "Table of thermodynamic parameters" is changed from "Breslauer et al. 1986" to "SantaLucia 1998" and "Salt correction formula" is changed from "Schildkraut and Lifson 1965" to "SantaLucia 1998". As a result, the default Tm values for your primers will be different from previous calculation. If you would like a different value for future use, you can save your custom parameters using "Save search parameters" at the top of the page with Tm = "Breslauer et al. 1986" and Salt correction = "Schildkraut and Lifson 1965" SNPsNo more than 1 or 2 SNPs in 5‘ end, no SNP in the 6nt closest to 3’endRepeatsNo more than 6 mono- or di- nucleotide repeats, or several shorted repeats broken by single alternative nucleotide, in the amplicon (visual check or repeat mask in amplicon BLAST)SpecificityPrimers are specific to intended target - consider homologous genes, transcript variants and pseudogenes Spacing Minimum of 5nt designed between each peak as they may end up closer due to CE. No less than 3nt ACTUAL space between peaks.Intron-spanningDesign primers on two separate exons that span a large intron. This is optional, but advised for samples where complete DNase digestion is not likely Shotgun ApproachProprietary Genes Information BLAST QC with NCBI(if possible)Redesign individual primers(as necessary)Primer 3 inputs SWhttp://frodo.wi.mit.edu/primer3/Order primers with universal tags and evaluate primers in vitro1. Enter FASTA sequence2. Enter the desired size range3. Click “Pick PrimersBLAST QC with NCBIhttp://blast.ncbi.nlm.nih.gov/Blast.cgiPaste FASTA Click BlastChecking SNP BLAST QC with NCBIhttp://blast.ncbi.nlm.nih.gov/Blast.cgi。