1. Essential Bioinformatics Resources for Designing PCR Primers and Oligos for Various Applications Please complete the workshop sign-in form. To help us develop bioinformatics workshops that are more relevant to your research, please take our online User Needs Survey if you have not done so, thanks! From the NML-Bioinformatics Web Site  Click the NML Support Requests under the “Support Request Section”  Click the “this online user needs survey” under the “Tell us how to serve your information needs better! Section”.NML-Bioinformatics Web Site NML Support Requeststhis online user needs survey

2. Essential Bioinformatics Resources for Designing PCR Primers and Oligos for Various Applications Yi-Bu Chen, Ph.D. Bioinformatics Specialist Norris Medical Library University of Southern California 323-442-3309 yibuchen@belen.hsc.usc.edu

3. Workshop Outline A.The General Rules for PCR Primer Design B.Resources for General Purpose PCR Primer Design C.Resources for SNPs and Genotyping PCR Applications D.Resources for Real-Time q-PCR Primer Design E.Resources for Site-Directed Mutagenesis PCR Primer Design F.Resources for PCR Primers/Oligos Quality Analysis G.Resources for Degenerate PCR Primer Design H.Resources for Microarray Probes Design I.Resources for Multiplex PCR Primer Design J.Resources Methylation PCR Primer Design

4.  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

5. Primers dictate the successfulness of a PCR Specificity? Proper annealing to the template?

6. Before you design your own primers – Don’t reinvent the wheels!

7. 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/rs ID?

8. 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  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

9. 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 application  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

10. General rules for primer design -- Melting temperature (T m )  T m 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: T m =2(A+T)+4(G+C) (suitable only for 18mer or shorter).  Optimal melting temperature  52°C-- 60°C  T m above 65°C should be generally avoided because of the potential for secondary annealing.  Higher T m (75°C-- 80°C) is recommended for amplifying high GC content targets.  Primer pair T m mismatch  Significant primer pair T m mismatch can lead to poor amplification  Desirable T m difference < 5°C between the primer pair

11. General rules for primer design -- Specificity and cross homology  Specificity  Determined primarily by primer length as well as the sequence  The adequacy of primer specificity is depended 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.

12. 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

13. 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;

14. 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.

15. General rules for primer design -- Annealing temperatures and other considerations  T a (Annealing temperature) vs. T m  T a is determined by the T m of both primers and amplicons: optimal T a =0.3 x T m (primer)+0.7 x T m (product)-25  General rule: T a is 5°C lower than T m  Higher T a 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  Consider nested PCR

16. http://www.hsls.pitt.edu/guides/genetics/obrc http://www.usc.edu/hsc/nml/lib-services/bioinformatics/index.html

17. http://search.hsls.pitt.edu/vivisimo/cgi-bin/query-meta?input-form=simple&v%3Asources=hsls_genetics_tools&v%3Aproject=molbio&query=primer*

18. Resources for General Purpose PCR Primer Design  Primer3  Primer3Plus  PrimerZ  PerlPrimer  Vector NTI Advantage 10

19. 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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1043858198/info

20. 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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1191263055/info

21. 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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1190992855/info

22. 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

23. 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_workshop.html More Info On Vector NTI Advance 10: http://www.usc.edu/hsc/nml/lib-services/bioinformatics/vector_nti_advance_10.html

24. PCR Primer Design Resources for SNPs and Genotyping Purposes  NCBI Probe Database  PrimerZ  MuPlex  SNPBox

25. 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

26. http://www.ncbi.nlm.nih.gov/sites/entrez?db=probe

27. 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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1190992855/info

28. 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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1135006767/info PCR Primer Design Tools for SNPs and Genotyping Purposes– MuPlex

29. Web Site: http://www.snpbox.org/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1097782408/info http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1097782408/info PCR Primer Design Tools for SNPs and Genotyping Purposes– SNPBox

30. Primer Design Resources for Real-time PCR  NCBI Probe Database  RTPrimerDB  Primer Bank  qPrimerDepot  PCR-QPPD  PerlPrimer

31. 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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1099597360/info

32. 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 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14654707

33. 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

34. 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

35. 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

36. 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

37. Resources for PCR Primer or Oligo Analysis  AutoDimer  IDT OligoAnalyzer 3.0  PUNS  NCBI BLAST  UCSC In-Silico PCR

38. 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

39. 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

40. 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

41. 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

42. http://genome.ucsc.edu/cgi-bin/hgPcr?db=mm9 Resources for PCR Primer Mapping – UCSC In-Silico PCR

43. http://www.bioinformatics.org/sms2/pcr_products.html http://www.bioinformatics.org/sms2/index.html Resources for PCR Primer Mapping/Amplicon Size – SMS Tool

44. 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

45. Primer Design Tools for Degenerate PCR  Primaclade  GeneFisher2  CODEHOP

46. 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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167846864/info

47. 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

48. 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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1118954832/info

49. Resources for Microarray Probe Design  NCBI Probe Database  OligoWiz 2.0  ROSO  YODA

50. Web Site: http://www.cbs.dtu.dk/services/OligoWiz2/ More Info: http://www.hsls.pitt.edu/guides/genetics/obrc/gene_expression/microarray_design_probes/URL111876 7631/info Resources for Microarray Probe Design –OligoWiz 2.0

51. 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

52. 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

53. Primer Design Tools for Multiplex PCR  MultiPLX  PrimerStation

54. 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 http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=AbstractPlus&list_uids=15598831

55. 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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1154793164/info

56. Primer Design Resources for Methylation PCR  MethPrimer  methBLAST and methPrimerDB  BiSearch  PerlPrimer

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 http://www.hsls.pitt.edu/guides/genetics/obrc/dna/pcr_oligos/URL1167846108/info

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

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

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

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. 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