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Essential Bioinformatics Resources
for Designing PCR Primers and
Oligos for Various Applications
A.Arputha Selvaraj
Workshop Outline
A. The General Rules for PCR Primer Design
B. Resources for General Purpose PCR Primer Design
C. Resource...
The Polymerase Chain Reaction (PCR) revolutionized life
sciences as it provides a sensitive, reliable, efficient, and
con...
Primers dictate the successfulness of a PCR
Specificity?
Proper
annealing to
the template?
Before you design your own primers –
Don’t reinvent the wheels!
Before you start designing primers –
Find and use the right resources!
What are the primers for?
 General purpose amplif...
After you have your primers designed –
Consider a second opinion!
Most likely your primers can be designed by
several dif...
General rules for primer design
-- Primer and amplicon length
Primer length determines the specificity and
significantly ...
General rules for primer design
-- Melting temperature (Tm)
 Tm is the temperature at which 50% of the DNA duplex
dissoci...
General rules for primer design
-- Specificity and cross homology
 Specificity
 Determined primarily by primer length as...
General rules for primer design
-- GC content; repeats and runs
Primer G/C content
 Optimal G/C content: 45-55%
 Common...
General rules for primer design
-- Primer secondary structures
 Hairpins
 Formed via intra-molecular interactions
 Nega...
General rules for primer design
-- GC clamp and max 3’ end stability
GC clamp
 Refers to the presence of G or C within t...
General rules for primer design
-- Annealing temperatures and other considerations
Ta (Annealing temperature) vs. Tm
 Ta...
http://www.hsls.pitt.edu/guides/genetics/obrc
http://www.usc.edu/hsc/nml/lib-services/bioinformatics/index.html
http://search.hsls.pitt.edu/vivisimo/cgi-bin/query-meta?input-form=molbio-simple&query=pcr+primer&v%3Asources=OBRC&v%3Apro...
Resources for General Purpose PCR Primer Design
 Primer3
 Primer3Plus
 PrimerZ
 PerlPrimer
 Vector NTI Advantage 10
General Purpose PCR Primer Design Tool– Primer3
Web Site:
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
More Inf...
General Purpose PCR Primer Design Tool– Primer3Plus
Web Site:
http://www.bioinformatics.nl/primer3plus
More Info:
http://w...
General Purpose PCR Primer Design Tool– PrimerZ
Web Site:
http://genepipe.ngc.sinica.edu.tw/primerz/beginDesign.do
More In...
General Purpose PCR Primer Design Tool –
PerlPrimer
Web Site:
http://perlprimer.sourceforge.net/index.html
PerlPrimer scre...
General Purpose PCR Primer Design Tool–
Vector NTI Advance 10
Web Site for NML Workshop:
http://www.usc.edu/hsc/nml/lib-se...
Primer Design Resources for Real-time PCR
 NCBI Probe Database
 RTPrimerDB
 Primer Bank
 qPrimerDepot
 PCR-QPPD
 Per...
Public PCR Primers/Oligo Probes Repository
– The NCBI Probe Database
Web Site:
Database Overview:
http://www.ncbi.nlm.nih....
http://www.ncbi.nlm.nih.gov/sites/entrez?db=probe
Resources for real time PCR– RTPrimerDB
Web Site:
http://medgen.ugent.be/rtprimerdb/
More Info:
http://www.hsls.pitt.edu/g...
Resources for real time PCR– Primer Bank
Web Site:
http://pga.mgh.harvard.edu/primerbank/
More Info:
http://www.ncbi.nlm.n...
Resources for real time PCR– qPrimerDepot
Web Site for Human Genes:
http://primerdepot.nci.nih.gov/
Web Site for Mouse Gen...
Resources for real time PCR– QPPD
Web Site:
http://web.ncifcrf.gov/rtp/GEL/primerdb/default.asp
More Info:
http://www.hsls...
Web Site:
http://perlprimer.sourceforge.net/index.html
PerlPrimer screenshots:
http://perlprimer.sourceforge.net/screensho...
Web Site:
http://www.bioinformatics.org/primerx/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/U...
Resources for PCR Primer or Oligo Analysis
 AutoDimer
 IDT OligoAnalyzer 3.0
 PUNS
 NCBI BLAST
 UCSC In-Silico PCR
Web Site:
http://www.cstl.nist.gov/div831/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm
More Info:
http://www.hsl...
Web Site:
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
Online Instruction:
http://www.idtdna.com/Analyzer/Ap...
Web Site:
http://okeylabimac.med.utoronto.ca/PUNS/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos...
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&MEGABLAST=on&BLAST_PROGRAMS=megaBlast&PAGE_TYP...
http://genome.ucsc.edu/cgi-bin/hgPcr?db=mm9
Resources for PCR Primer Mapping
– UCSC In-Silico PCR
http://www.bioinformatics.org/sms2/pcr_products.html
http://www.bioinformatics.org/sms2/index.html
Resources for PCR Prime...
Please evaluate this workshop to help me improving
future presentations:
http://www.zoomerang.com/survey.zgi?p=WEB2277FTDR...
Primer Design Tools for Multiplex PCR
 MultiPLX
 PrimerStation
Primer Design Tools for Multiplex PCR–
MultiPLX
Web Site:
http://bioinfo.ebc.ee/multiplx/
More Info:
http://www.ncbi.nlm.n...
Primer Design Tools for Multiplex PCR–
PrimerStation
Web Site:
http://ps.cb.k.u-tokyo.ac.jp/index.html
More Info:
http://w...
Resources for Microarray Probe Design
 NCBI Probe Database
 OligoWiz 2.0
 ROSO
 YODA
Web Site:
http://www.cbs.dtu.dk/services/OligoWiz2/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/gene_expressi...
Web Site:
http://pbil.univ-lyon1.fr/roso/Home.php
More Info:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDe...
Web Site:
http://pathport.vbi.vt.edu/YODA/
More Info:
http://bioinformatics.oxfordjournals.org/cgi/content/full/21/8/1365
...
PCR Primer Design Resources for SNPs and Genotyping Purposes
 NCBI Probe Database
 PrimerZ
 MuPlex
 SNPBox
Public PCR Primers/Oligo Probes Repository
– The NCBI Probe Database
Web Site:
Database Overview:
http://www.ncbi.nlm.nih....
PCR Primer Design Resources for SNPs and Genotyping Purposes
– PrimerZ
Web Site:
http://genepipe.ngc.sinica.edu.tw/primerz...
Web Site:
http://genomics14.bu.edu:8080/MuPlex/MuPlex.html
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pc...
Web Site:
http://www.snpbox.org/
More Info:
http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1097782408/inf...
Primer Design Tools for Degenerate PCR
 Primaclade
 GeneFisher2
 CODEHOP
Primer Design Tools for Degenerate PCR– Primaclade
Web Site:
http://www.umsl.edu/services/kellogg/primaclade.html
More Inf...
Primer Design Tools for Degenerate PCR– GeneFisher2
Web Site:
http://bibiserv.techfak.uni-bielefeld.de/genefisher2/
More I...
Primer Design Tools for Degenerate PCR– CODEHOP
Web Site:
http://blocks.fhcrc.org/codehop.html
More Info:
http://www.hsls....
Primer Design Resources for Methylation PCR
 MethPrimer
 methBLAST and methPrimerDB
 BiSearch
 PerlPrimer
Primer Design Resources for Methylation PCR–
MethPrimer
Web Site:
http://www.urogene.org/methprimer/
More Info:
http://www...
Primer Design Tools for Methylation PCR–
methBLAST and methPrimerDB
methPrimerDB Web Site:
http://medgen.ugent.be/methprim...
Primer Design Tools for Methylation PCR–
BiSearch
Web Site:
http://bisearch.enzim.hu/
More Info:
http://www.ncbi.nlm.nih.g...
Primer Design Tools for Methylation PCR–
PerlPrimer
Web Site:
http://perlprimer.sourceforge.net/index.html
PerlPrimer scre...
Please evaluate this workshop to help me improving
future presentations:
http://www.zoomerang.com/survey.zgi?p=WEB2277FTDR...
Useful web sites for design degenerate PCR primers
http://boneslab.bio.ntnu.no/degpcrshortguide.htm
http://info.med.yale.e...
Thank YouThank You
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Transcript of "PCR DNA PRIMER"

  1. 1. Essential Bioinformatics Resources for Designing PCR Primers and Oligos for Various Applications A.Arputha Selvaraj
  2. 2. Workshop Outline A. The General Rules for PCR Primer Design B. Resources for General Purpose PCR Primer Design C. Resources for Real-Time q-PCR Primer Design D. Resources for Site-Directed Mutagenesis PCR Primer Design E. Resources for PCR Primers/Oligos Quality Analysis F. Resources for Multiplex PCR Primer Design G. Resources for Microarray Probes Design H. Resources for SNPs and Genotyping PCR Applications I. Resources for Degenerate PCR Primer Design J. Resources Methylation PCR Primer Design
  3. 3. The Polymerase Chain Reaction (PCR) revolutionized life sciences as it provides a sensitive, reliable, efficient, and convenient means of amplifying relatively large quantities of DNA Invented in 1983 by Kary Mullis, who won a Nobel Prize 1993 The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus, discovered in hot springs. The primary materials used in PCR: - DNA nucleotides: the building blocks for the new DNA - Template DNA: the DNA sequence that you want to amplify - Primers: single-stranded short DNA (16--50 nucleotides long) that are complementary to a short region on either end of the template DNA - DNA polymerase: a heat stable enzyme that catalyzes the synthesis of new DNA PCR: the technology that changed the world we knew
  4. 4. Primers dictate the successfulness of a PCR Specificity? Proper annealing to the template?
  5. 5. Before you design your own primers – Don’t reinvent the wheels!
  6. 6. Before you start designing primers – Find and use the right resources! What are the primers for?  General purpose amplification?  SNPs detection/validation?  Methylation study?  Real-time PCR?  Microarray probes?  Degenerate PCR?  Multiplex PCR? What do you have to begin with?  Single DNA/protein sequence?  Multiple DNA/protein sequence files?  GenBank ID/Gene ID/Gene Symbol/rsSNP ID?
  7. 7. After you have your primers designed – Consider a second opinion! Most likely your primers can be designed by several different software Different software may vary significantly in:  Concepts and overall approaches  Designing criteria and default settings  Comprehensiveness  Usability  Accessibility and speed Consider a second opinion when  You are new to such design task/application  You don’t have a lot of confidence in the initial result
  8. 8. General rules for primer design -- Primer and amplicon length Primer length determines the specificity and significantly affect its annealing to the template  Too short -- low specificity, resulting in non-specific amplification  Too long -- decrease the template-binding efficiency at normal annealing temperature due to the higher probability of forming secondary structures such as hairpins. Optimal primer length  18-24 bp for general applications  30-35 bp for multiplex PCR Optimal amplicon size  300-1000 bp for general application, avoid > 3 kb  50-150 bp for real-time PCR, avoid > 400 bp
  9. 9. General rules for primer design -- Melting temperature (Tm)  Tm is the temperature at which 50% of the DNA duplex dissociates to become single stranded  Determined by primer length, base composition and concentration.  Also affected by the salt concentration of the PCR reaction mix  Working approximation: Tm=2(A+T)+4(G+C) (suitable only for 18mer or shorter).  Optimal melting temperature  52°C-- 60°C  Tm above 65°C should be generally avoided because of the potential for secondary annealing.  Higher Tm (75°C-- 80°C) is recommended for amplifying high GC content targets.  Primer pair Tm mismatch  Significant primer pair Tm mismatch can lead to poor amplification
  10. 10. General rules for primer design -- Specificity and cross homology  Specificity  Determined primarily by primer length as well as sequence  The adequacy of primer specificity is dependent on the nature of the template used in the PCR reaction.  Cross homology  Cross homology may become a problem when PCR template is genomic DNA or consists of mixed gene fragments.  Primers containing highly repetitive sequence are prone to generate non- specific amplicons when amplifying genomic DNA.  Avoid non-specific amplification  BLASTing PCR primers against NCBI non-redundant sequence database is a common way to avoid designing primers that may amplify non-targeted homologous regions.  Primers spanning intron-exon boundaries to avoid non-specific amplification of gDNA due to cDNA contamination.  Primers spanning exon-exon boundaries to avoid non-specific amplification cDNA due to gDNA contamination.
  11. 11. General rules for primer design -- GC content; repeats and runs Primer G/C content  Optimal G/C content: 45-55%  Common G/C content range: 40-60% Runs (single base stretches)  Long runs increases mis-priming (non-specific annealing) potential  The maximum acceptable number of runs is 4 bp Repeats (consecutive di-nucleotide)  Repeats increases mis-priming potential  The maximum acceptable number of repeats is 4 di- nucleotide
  12. 12. General rules for primer design -- Primer secondary structures  Hairpins  Formed via intra-molecular interactions  Negatively affect primer-template binding, leading to poor or no amplification  Acceptable ΔG (free energy required to break the structure): >-2 kcal/mol for 3’end hairpin; >-3 kcal/mol for internal hairpin;  Self-Dimer (homodimer)  Formed by inter-molecular interactions between the two same primers  Acceptable ΔG: >-5 kcal/mol for 3’end self-dimer; >-6 kcal/mol for internal self-dimer;  Cross-Dimer (heterodimer)  Formed by inter-molecular interactions between the sense and antisense primers  Acceptable ΔG: >-5 kcal/mol for 3’end cross-dimer; >-6 kcal/mol for internal cross-dimer;
  13. 13. General rules for primer design -- GC clamp and max 3’ end stability GC clamp  Refers to the presence of G or C within the last 4 bases from the 3’ end of primers  Essential for preventing mis-priming and enhancing specific primer-template binding  Avoid >3 G’s or C’s near the 3’ end Max 3’end stability  Refers to the maximum ΔG of the 5 bases from the 3’end of primers.  While higher 3’end stability improves priming efficiency, too higher stability could negatively affect specificity because of 3’-terminal partial hybridization induced non- specific extension.  Avoid ΔG < -9.
  14. 14. General rules for primer design -- Annealing temperatures and other considerations Ta (Annealing temperature) vs. Tm  Ta is determined by the Tm of both primers and amplicons: optimal Ta=0.3 x Tm(primer)+0.7 x Tm(product)-25  General rule: Ta is 5°C lower than Tm  Higher Ta enhances specific amplification but may lower yields  Crucial in detecting polymorphisms Primer location on template  Dictated by the purpose of the experiment  For detection purpose, section towards 3’ end may be preferred. When using composite primers  Initial calculations and considerations should emphasize on the template-specific part of the primers
  15. 15. http://www.hsls.pitt.edu/guides/genetics/obrc http://www.usc.edu/hsc/nml/lib-services/bioinformatics/index.html
  16. 16. http://search.hsls.pitt.edu/vivisimo/cgi-bin/query-meta?input-form=molbio-simple&query=pcr+primer&v%3Asources=OBRC&v%3Aproject=molbio
  17. 17. Resources for General Purpose PCR Primer Design  Primer3  Primer3Plus  PrimerZ  PerlPrimer  Vector NTI Advantage 10
  18. 18. General Purpose PCR Primer Design Tool– Primer3 Web Site: http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1043858198/info Name Primer3 -- an online tool for PCR primer design Type Web-based software Key Functions Design PCR primers and hybridization probes. Publication Info Methods Mol Biol 2000 Times Cited 823 Pros The original and most widely used PCR primer design program; uses sequence as input; a huge number of options for customizing primer design; Cons busy interface; Note In OBRC; the program has been widely adopted by many primer design software. YiBu’s Rating 4 out of 5
  19. 19. General Purpose PCR Primer Design Tool– Primer3Plus Web Site: http://www.bioinformatics.nl/primer3plus More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1191263055/info Name Primer3Plus, an enhanced web interface to Primer3 Type Web-based software Key Functions Design PCR primers for a given DNA sequence. Publication Info NAR 2007 Times Cited N/A Pros Uses sequences or sequence file as input; a huge number of configuration options; automates specific tasks such as designing primers for cloning or step- wise sequencing; primers can be sent to an order form; clean, intuitive and well organized interface; Cons Note in OBRC. It is an updated, task-oriented web-interface to the original Primer3. YiBu’s Rating 4.5 out of 5
  20. 20. General Purpose PCR Primer Design Tool– PrimerZ Web Site: http://genepipe.ngc.sinica.edu.tw/primerz/beginDesign.do More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1190992855/info Name PrimerZ -- streamlined primer design for promoters, exons and human/mouse SNPs Type Web-based software Key Functions Design PCR primers for promoters, exons, and human/mouse SNPs Publication Info Nucleic Acid Research 2007 Times Cited 0 (too new) Pros Uses gene name, Ensembl ID, rs# as input; settings for amplicon region and length, as well as PCR product sizes; allow batch rsSNP processing; frequently updated; offers many advance design settings; interactive results output Cons reported successful rate over 70%; only for human and mouse Note built on Primer3; in OBRC. YiBu’s Rating 4.5 out of 5
  21. 21. General Purpose PCR Primer Design Tool – PerlPrimer Web Site: http://perlprimer.sourceforge.net/index.html PerlPrimer screenshots: http://perlprimer.sourceforge.net/screenshots.html More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167845497/info Name PerlPrimer -- cross-platform, graphical primer design for standard, bisulphite and real-time PCR Type Desktop software (for Windows, Linux and Mac OS-X) Key Functions Design primers for standard, bisulphite and real-time PCR, and sequencing. Publication Info Bioinformatics 2004 Times Cited 8 Pros Cross-platform; versatile applications; retrieving sequences from Ensembl as input; QPCR primer design without manual intron-exon boundary entry; BLAST search primers; primer pair quality analysis; ORF and CpG island detection; Cons Requires local installation of the software; automatic sequence retrieval only through Esemble (selected eukaryotic genomes) not NCBI (more comprehensive). Note In OBRC YiBu’s Rating 4 out of 5
  22. 22. General Purpose PCR Primer Design Tool– Vector NTI Advance 10 Web Site for NML Workshop: http://www.usc.edu/hsc/nml/lib-services/bioinformatics/vector_nti_advance_10_wo More Info On Vector NTI Advance 10: http://www.usc.edu/hsc/nml/lib-services/bioinformatics/vector_nti_advance_10.html Name Vector NTI Advance 10 -- design primers and oligos for regular/multiplex PCR, sequencing, hybridization. Type Commercial Desktop software (free to nonprofit users) Key Functions Design PCR primers and oligos for routine molecular applications Publication Info N/A Times Cited N/A Pros Uses user sequence, multiple DNA sequence alignment as input; commercial quality features/functions; integrated with other Vector NTI applications; many design settings; analyzes and ranks primer quality; Cons requires software installation/licensing; Note NML offers workshop and tutorials; in OBRC. YiBu’s Rating 4 out of 5
  23. 23. Primer Design Resources for Real-time PCR  NCBI Probe Database  RTPrimerDB  Primer Bank  qPrimerDepot  PCR-QPPD  PerlPrimer
  24. 24. Public PCR Primers/Oligo Probes Repository – The NCBI Probe Database Web Site: Database Overview: http://www.ncbi.nlm.nih.gov/genome/probe/doc/Overview.shtml Database Query Tips: http://www.ncbi.nlm.nih.gov/genome/probe/doc/QueryTips.shtml Name NCBI Probe Database Type Web-based database Key Functions Search for documented PCR primers and oligo probes for genotyping, gene expression, SNP discovery, gene silencing and genome mapping applications. Publication Info Unpublished Times Cited n/a Pros The largest database of its kind; results including information on reagent distributors, probe effectiveness, and computed sequence similarities; Entrez allows search to be limited by applications, probe type, and model organism; Cons n/a Note YiBu’s Rating 4.5 out of 5
  25. 25. http://www.ncbi.nlm.nih.gov/sites/entrez?db=probe
  26. 26. Resources for real time PCR– RTPrimerDB Web Site: http://medgen.ugent.be/rtprimerdb/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1099597360/info Name RTPrimerDB: the real-time PCR primer and probe database Type Database Key Functions Search for validated primers and probes used in real-time PCR assays employing popular chemistries (SYBR Green I, Taqman, Hybridisation Probes, Molecular Beacon); map primers/probes onto different transcript variants Publication Info Nucleic Acids Research 2003, 2006 (update) Times Cited 62 Pros Primers/probes experimentally validated; search with gene name/symbol, Entrez/Ensembl Gene identifier, SNP ID, or oligo sequence; queries can be limited to a specific application (gene expression, DNA copy number, SNP detection, mutation analysis, fusion gene) or organisms (20); results linked to PubMed record and BLAST; frequently updated; Cons Gene expression assay viewer requires Adobe SVG viewer plug-in, and only available for human, mouse, rat. Note In OBRC; 3845 real-time PCR assays for 2373 genes as of Nov. 2007. YiBu’s Rating 4.8 out of 5
  27. 27. Resources for real time PCR– Primer Bank Web Site: http://pga.mgh.harvard.edu/primerbank/ More Info: http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14654707 Name A PCR primer bank for quantitative gene expression analysis Type Web-based database Key Functions Search for pre-designed transcript-specific PCR primers for genome-scale real time PCR assay Publication Info Nucleic Acids Research 2003 Times Cited 67 Pros Searchable by GenBank Accession number, gene ID/symbol, keywords etc.; design algorithm extensively tested and validated with success rate of 82.6%; results output containing positions of primers and amplicons in the sequence context of the queried gene Cons Only for human and mouse genes; algorithm not designed to span introns; no graphic display; Note Contains 306,800 real-time PCR primers for 33741 human genes and 27681 mouse genes as of Nov. 2007 YiBu’s Rating 4.8 out of 5
  28. 28. Resources for real time PCR– qPrimerDepot Web Site for Human Genes: http://primerdepot.nci.nih.gov/ Web Site for Mouse Genes: http://mouseprimerdepot.nci.nih.gov/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1174922412/info Name qPrimerDepot -- a primer database for quantitative real time PCR Type Web-based database Key Functions Provides optimized qRT-PCR primers for all human/mouse RefSeq genes; Publication Info NAR 2005 Times Cited 19 Pros Uses GenBank RefSeq ID or gene name as input; primers are designed to amplify desired templates under unified annealing temperature; avoids genomic DNA contamination (for intron-bearing genes); simple user interface; Validation results show 70-94% successful rate; Cons Results output without sequence context display of primer/amplicons positions Note In OBRC; built on Primer3; YiBu’s Rating 4.5 out of 5
  29. 29. Resources for real time PCR– QPPD Web Site: http://web.ncifcrf.gov/rtp/GEL/primerdb/default.asp More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1152117830/info Name QPPD -- Quantitative PCR Primer Database for human and mouse Type Web-based database Key Functions Search for published quantitative/real time RT-PCR primer and probes for studying of human and mouse gene expression. Publication Info Unpublished Times Cited n/a Pros All primers were experimentally validated with literature references; uses gene name as input; searches can be limited by different assay types; output includes graphic display of primers/amplicons positions and sizes. Cons Only human and mouse genes available; no user documentations and database stats. Note In OBRC YiBu’s Rating 4 out of 5
  30. 30. Web Site: http://perlprimer.sourceforge.net/index.html PerlPrimer screenshots: http://perlprimer.sourceforge.net/screenshots.html More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167845497/info Resources for real time PCR– PerlPrimer Name PerlPrimer -- cross-platform, graphical primer design for standard, bisulphite and real-time PCR Type Desktop software (for Windows, Linux and Mac OS-X) Key Functions Design primers for standard, bisulphite and real-time PCR, and sequencing. Publication Info Bioinformatics 2004 Times Cited 8 Pros Cross-platform; versatile applications; retrieving sequences from Ensembl as input; QPCR primer design without manual intron-exon boundary entry; BLAST search primers; primer pair quality analysis; ORF and CpG island detection; Cons Requires local installation of the software; automatic sequence retrieval only through Esemble (selected eukaryotic genomes) not NCBI (more comprehensive). Note In OBRC YiBu’s Rating 4 out of 5
  31. 31. Web Site: http://www.bioinformatics.org/primerx/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1175091818/info Resources for Site-Directed Mutagenesis PCR – PrimerX Name PrimerX -- Automated design of mutagenic primers for site-directed mutagenesis Type Web-based software Key Functions Design mutagenic primers for site-directed mutagenesis. Publication Info unpublished Times Cited n/a Pros Uses either DNA or protein sequences as input; results can be customized for three different commercial mutagenesis kits; straightforward user interface, Cons No data on evaluation and user feedback; Note In OBRC YiBu’s Rating 4 out of 5
  32. 32. Resources for PCR Primer or Oligo Analysis  AutoDimer  IDT OligoAnalyzer 3.0  PUNS  NCBI BLAST  UCSC In-Silico PCR
  33. 33. Web Site: http://www.cstl.nist.gov/div831/strbase/AutoDimerHomepage/AutoDimerProgramHomepage.htm More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1154964478/info Resources for PCR Primer or Oligo Analysis – AutoDimer Name AutoDimer -- a screening tool for primer-dimer and hairpin structures Type Web-based or desktop software Key Functions Rapidly screen PCR primers for primer-dimer and hairpin interactions in short DNA oligomers (< 30 nucleotides) Publication Info Biotechniques 2004 Times Cited 17 Pros Suited for screening multiplex PCR primers; output has alignment; Cons Requires manually formatted primers file as input; crude web layout; the web- based version is limited 100 sequences/run and maximum oligomer length of 75 nucleotides or less. Note In OBRC YiBu’s Rating 4 out of 5
  34. 34. Web Site: http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/ Online Instruction: http://www.idtdna.com/Analyzer/Applications/Instructions/Default.aspx?AnalyzerInstructions=true Resources for PCR Primer or Oligo Analysis –IDT OligoAnalyzer 3.0 Name IDT OligoAnalyzer 3.0 Type Web-based software Key Functions Analyze primer/oligo sequence and structure. Publication Info Unpublished Times Cited n/a Pros Includes analysis of hairpins, self-dimer, heterodimer; customizable primer/oligo and salt concentrations; many options for various sequence modifications; direct submission for NCBI BLAST; allows direct order of primer/oligo; Cons No evaluation data. Note YiBu’s Rating 4 out of 5
  35. 35. Web Site: http://okeylabimac.med.utoronto.ca/PUNS/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1175092441/info Resources for PCR Primer Specificity Analysis – PUNS Name PUNS -- Transcriptomic- and genomic-in silico PCR for enhanced primer design Type Web-based or desktop software (Windows, Linux) Key Functions Use in silico PCR to verify primer specificity by comparing the primers against the entire transcriptome/genome and looking for alternate binding and potential alternate amplicons. Particularly suited for the identification of highly selective primers for quantitative microarray validation; Publication Info Bioinformatics 2004 Times Cited 4 Pros Focuses on primer specificity analysis; capable of design cross-species primers; uses pre-designed primers as input; good online documentation Cons For specificity check against transcriptome, works only with model organisms whose transcriptome info are available in UniGene; requires special input file format; unnecessary complicated interface; Note in OBRC YiBu’s Rating 4 out of 5
  36. 36. http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&MEGABLAST=on&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on Resources for PCR Primer Specificity Analysis – NCBI BLAST
  37. 37. http://genome.ucsc.edu/cgi-bin/hgPcr?db=mm9 Resources for PCR Primer Mapping – UCSC In-Silico PCR
  38. 38. http://www.bioinformatics.org/sms2/pcr_products.html http://www.bioinformatics.org/sms2/index.html Resources for PCR Primer Mapping/Amplicon Size – SMS Tool
  39. 39. Please evaluate this workshop to help me improving future presentations: http://www.zoomerang.com/survey.zgi?p=WEB2277FTDR3AJ Have questions or comments about this workshop? Please contact: Yi-Bu Chen, Ph.D. Bioinformatics Specialist Norris Medical Library University of Southern California 323-442-3309 yibuchen@belen.hsc.usc.edu
  40. 40. Primer Design Tools for Multiplex PCR  MultiPLX  PrimerStation
  41. 41. Primer Design Tools for Multiplex PCR– MultiPLX Web Site: http://bioinfo.ebc.ee/multiplx/ More Info: http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15598831 Name MultiPLX -- automatic grouping and evaluation of PCR primers Type Web-based or desktop software (Windows, Linux) Key Functions Automatic grouping large number of PCR primers pairs based on analysis of primers properties and compatibilities. Publication Info Bioinformatics 2005 Times Cited 2 Pros Capable of analyzing thousands of primers; Cons Input requires specially formatted primer file; poor online documentation; Note YiBu’s Rating 3.5 out of 5
  42. 42. Primer Design Tools for Multiplex PCR– PrimerStation Web Site: http://ps.cb.k.u-tokyo.ac.jp/index.html More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1154793164/info Name PrimerStation -- a highly specific multiplex genomic PCR primer design server for the human genome Type Web-based software Key Functions Design highly specific and accurate multiplex genomic PCR primer for human genome. Publication Info NAR 2006 Times Cited 0 (too new) Pros Uses multiple Genbank RefSeq ID or chromosomal coordinates as inputs; allows exon-only amplification; options to avoid SNPs region or CA-repeats; configurable settings for primer design; Cons Only for human genome; Note In OBRC YiBu’s Rating 4.5 out of 5
  43. 43. Resources for Microarray Probe Design  NCBI Probe Database  OligoWiz 2.0  ROSO  YODA
  44. 44. Web Site: http://www.cbs.dtu.dk/services/OligoWiz2/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/gene_expression/microarray_design_probes/URL111876763 Resources for Microarray Probe Design –OligoWiz 2.0 Name OligoWiz 2.0 -- integrating sequence feature annotation into the design of microarray probes Type Web-based client-server solution Key Functions Perform intelligent design of oligonucleotides against a given set of target sequences for DNA microarray application. Publication Info NAR 2005 Times Cited 8 Pros The input sequence file can be in FASTA or annotation containing tab format; integrated species database for reducing or eliminating cross-hybridizations; in addition to probe selection according to a series of probe quality parameters, cross-hybridization, Tm, position in transcript, probe folding and low- complexity, the program facilitates automatic placement of probes relative to the sequence annotation; evaluation study shows consistent hybridization results; decent online user guide; Cons Requires download/install Java-based Graphic User Interface Note In OBRC YiBu’s Rating 4 out of 5
  45. 45. Web Site: http://pbil.univ-lyon1.fr/roso/Home.php More Info: http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14734320 Resources for Microarray Probe Design –ROSO Name ROSO -- Optimizing oligonucleotide probes for microarrays Type Web-based software Key Functions Design optimal oligonucleotide probe sets for microarrays. Publication Info Bioinformatics 2004 Times Cited 23 Pros Uses FASTA formatted file for both targeted cDNA sequences and a facultative external file containing sequences to be avoided in cross- hybridization; the facultative files are provided for each model organisms, customized facultative can be uploaded; settings for hybridization conditions and probe secondary structures; Cons No data on evaluation and user feedback; Note YiBu’s Rating 4 out of 5
  46. 46. Web Site: http://pathport.vbi.vt.edu/YODA/ More Info: http://bioinformatics.oxfordjournals.org/cgi/content/full/21/8/1365 Resources for Microarray Probe Design –YODA Name YODA -- selecting signature oligonucleotides Type Desktop software Key Functions Select signature sequences for microarray and other applications Publication Info Bioinformatics 2004 Times Cited 15 Pros Cross-platform (Windows, Mac OS X, Linux); relies on a custom sequence similarity search algorithm instead of BLAST to minimize false negatives; supports multiple probe design goals including single-genome, multiple- genome, pathogen-host and species/strain-identification; many configurable parameters; uses FASTA formatted files for both targeted and avoided sequences. Cons Requires download and installation on local PC; no data on evaluation and user feedback; Note YiBu’s Rating 4 out of 5
  47. 47. PCR Primer Design Resources for SNPs and Genotyping Purposes  NCBI Probe Database  PrimerZ  MuPlex  SNPBox
  48. 48. Public PCR Primers/Oligo Probes Repository – The NCBI Probe Database Web Site: Database Overview: http://www.ncbi.nlm.nih.gov/genome/probe/doc/Overview.shtml Database Query Tips: http://www.ncbi.nlm.nih.gov/genome/probe/doc/QueryTips.shtml Name NCBI Probe Database Type Web-based database Key Functions Search for documented PCR primers and oligo probes for genotyping, gene expression, SNP discovery, gene silencing and genome mapping applications. Publication Info Unpublished Times Cited n/a Pros The largest database of its kind; results including information on reagent distributors, probe effectiveness, and computed sequence similarities; Entrez allows search to be limited by applications, probe type, and model organism; Cons n/a Note YiBu’s Rating 4.5 out of 5
  49. 49. PCR Primer Design Resources for SNPs and Genotyping Purposes – PrimerZ Web Site: http://genepipe.ngc.sinica.edu.tw/primerz/beginDesign.do More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1190992855/info Name PrimerZ -- streamlined primer design for promoters, exons and human/mouse SNPs Type Web-based software Key Functions Design PCR primers for promoters, exons, and human/mouse SNPs Publication Info Nucleic Acid Research 2007 Times Cited 0 (too new) Pros Uses gene name, Ensembl ID, rs# as input; settings for amplicon region and length, as well as PCR product sizes; allow batch rsSNP processing; frequently updated; offers many advance design settings; interactive results output Cons reported successful rate over 70%. Note built on Primer3; in OBRC. YiBu’s Rating 4.5 out of 5
  50. 50. Web Site: http://genomics14.bu.edu:8080/MuPlex/MuPlex.html More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1135006767/info PCR Primer Design Tools for SNPs and Genotyping Purposes– MuPlex Name MuPlex -- multi-objective multiplex PCR assay design Type Web-based software Key Functions Design primers for high-throughput multiplex PCR assays, including genotyping. Publication Info NAR 2005 Times Cited 7 Pros Input uses FASTA sequence/file; sequence with SNP brackets; allows masked region; results of assay solutions emailed; many design options; Cons No evaluation data Note In OBRC; build on Primer3 YiBu’s Rating 4 out of 5
  51. 51. Web Site: http://www.snpbox.org/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1097782408/info PCR Primer Design Tools for SNPs and Genotyping Purposes– SNPBox Name SNPbox: a modular software package for large-scale primer design Type Web-based software Key Functions Design PCR primers for large-scale amplification and sequencing projects aimed at constructing single nucleotide polymorphisms maps; design of primer sets for mutation analysis, STR marker genotyping and microarray oligos. Publication Info NAR 2004/Bioinformatics 2005 Times Cited 5 Pros Uses GenBank ID or FASTA sequence file as input; no prior knowledge of rsID needed; many design options; offers SNP; Exon and Saturation module; Cons Interface not well designed; inconsistent server processing speed; requires Adobe SVG viewer web browser plug-in for interactive results visualization. Note In OBRC; build on Primer3; YiBu’s Rating 3.5 out of 5
  52. 52. Primer Design Tools for Degenerate PCR  Primaclade  GeneFisher2  CODEHOP
  53. 53. Primer Design Tools for Degenerate PCR– Primaclade Web Site: http://www.umsl.edu/services/kellogg/primaclade.html More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167846864/info Name Primaclade -- a flexible tool to find conserved PCR primers across multiple species Type Web-based software Key Functions Design degenerated PCR primers based on conserved regions from a multiple species nucleotide alignment file Publication Info Bioinformatics 2005 Times Cited 9 Pros Uses a multiple species nucleotide alignment file as input; accepts several alignment format; Cons May not work well when sequences in the alignment are > 29% divergent; does not use IUPAC codes in the input alignment; last updated in 2004 Note Built on Primer3; in OBRC YiBu’s Rating 4 out of 5
  54. 54. Primer Design Tools for Degenerate PCR– GeneFisher2 Web Site: http://bibiserv.techfak.uni-bielefeld.de/genefisher2/ More Info: http://www.ncbi.nlm.nih.gov/sites/entrez? Db=pubmed&Cmd=ShowDetailView&TermToSearch=8877506 Name GeneFisher2 – an interactive tool for designing degenerate PCR primers flexible tool to find conserved PCR primers across multiple species Type Web-based software Key Functions Design degenerated PCR primers based on conserved regions among multiple DNA or protein sequences Publication Info Proc Int Conf Intell Syst Mol Biol. 1996 Times Cited 38 Pros Uses but does not require pre-aligned sequences; integrated alignment tools to align starting sequences; accepts both DNA and protein sequences; Cons Only accepts FASTA format alignment; no evaluation data; Note Built on Primer3; YiBu’s Rating 4 out of 5
  55. 55. Primer Design Tools for Degenerate PCR– CODEHOP Web Site: http://blocks.fhcrc.org/codehop.html More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1118954832/info Name CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design Type Web-based software Key Functions Design degenerate PCR primers based on multiple protein sequences alignments Publication Info Nucleic Acids Research 2003 Times Cited 37 Pros Widely cited with many successful applications; settings for genetic code and codon usage; Cons Requires local multiple alignment as input and must be in Blocks Database format; Note In OBRC YiBu’s Rating 4 out of 5
  56. 56. Primer Design Resources for Methylation PCR  MethPrimer  methBLAST and methPrimerDB  BiSearch  PerlPrimer
  57. 57. Primer Design Resources for Methylation PCR– MethPrimer Web Site: http://www.urogene.org/methprimer/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167846108/info Name MethPrimer: designing primers for methylation PCRs Type Web-based software Key Functions Design primers for methylation-specific, bisulfite-sequencing PCR, or bisulfite- restriction PCR assays. Publication Info Bioinformatics 2002 Times Cited 123 Pros Widely used; uses DNA sequence in any format as input; no need to modify sequences; very simple interface; settings for primers; output includes graphics and sequence alignment; fast processing Cons n/a Note Build on Primer3; in OBRC; YiBu’s Rating 4.5 out of 5
  58. 58. Primer Design Tools for Methylation PCR– methBLAST and methPrimerDB methPrimerDB Web Site: http://medgen.ugent.be/methprimerdb/ methBLAST Web Site: http://medgen.ugent.be/methBLAST/ More Info: http://www.biomedcentral.com/1471-2105/7/496 Name methBLAST and methPrimerDB: web-tools for PCR based methylation analysis Type Web-based software Key Functions methPrimerDB for validated PCR primers for DNA methylation analysis; methBLAST for in silico assessment of primer specificity in PCR based methylation assays. Publication Info BMC Bioinformatics 2006 Times Cited 0 (too new) Pros Search validated primers by gene name with organism limit; in silico PCR using bisulfite converted DNA as input; Cons Only 243 primer sets available in the methPrimerDB (as of Oct. 2007) Note in OBRC; from the same group of MethPrimer YiBu’s Rating 4 out of 5
  59. 59. Primer Design Tools for Methylation PCR– BiSearch Web Site: http://bisearch.enzim.hu/ More Info: http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15653630 Name BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes Type Web-based software Key Functions Design primers for both bisulfite-treated and native DNA templates. Publication Info NAR 2005, BMC Bioinformatics 2006, Methods Mol Biol 2007 Times Cited 8 Pros Uses plain text formatted sequence as input; integrated high-speed ePCR tool for fast detection of mispriming sites and alternative PCR products in cDNA libraries and native or bisulfite-treated genomes; tools for calculating primer Tm and scores; configurable designing/scoring parameters; fast server; Cons ePCR only available for 4 native/bisufite-treated mammalian genomes. Note YiBu’s Rating 4.5 out of 5
  60. 60. Primer Design Tools for Methylation PCR– PerlPrimer Web Site: http://perlprimer.sourceforge.net/index.html PerlPrimer screenshots: http://perlprimer.sourceforge.net/screenshots.html More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167845497/info Name PerlPrimer -- cross-platform, graphical primer design for standard, bisulphite and real-time PCR Type Desktop software (for Windows, Linux and Mac OS-X) Key Functions Design primers for standard, bisulphite and real-time PCR, and sequencing. Publication Info Bioinformatics 2004 Times Cited 8 Pros Cross-platform; versatile applications; retrieving sequences from Ensembl as input; QPCR primer design without manual intron-exon boundary entry; BLAST search primers; primer pair quality analysis; ORF and CpG island detection; Cons Requires local installation of the software; automatic sequence retrieval only through Esemble (selected eukaryotic genomes) not NCBI (more comprehensive). Note In OBRC YiBu’s Rating 4 out of 5
  61. 61. Please evaluate this workshop to help me improving future presentations: http://www.zoomerang.com/survey.zgi?p=WEB2277FTDR3AJ Have questions or comments about this workshop? Please contact: Yi-Bu Chen, Ph.D. Bioinformatics Specialist Norris Medical Library University of Southern California 323-442-3309 yibuchen@belen.hsc.usc.edu
  62. 62. Useful web sites for design degenerate PCR primers http://boneslab.bio.ntnu.no/degpcrshortguide.htm http://info.med.yale.edu/mbb/koelle/protocols/protocol_degenerate_PCR.html http://www.mcb.uct.ac.za//pcroptim.htm#Degenerate http://www.protocol-online.org/prot/Molecular_Biology/PCR/Degenerate_PCR/ http://cgat.ukm.my/protease/degpcr.html
  63. 63. Thank YouThank You
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