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 Multiplex PCR Primer Design E.Resources Methylation PCR Primer Design F.Resources for Degenerate PCR Primer Design G.Resources for Real-Time q-PCR Primer Design H.Resources for Site-Directed Mutagenesis PCR Primer Design I.Resources for PCR Primers/Oligos Quality Analysis

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 to have 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  MPrime ( server unavailable as of Jan. 2008)  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– MPrime Web Site: http://kbrin.a-bldg.louisville.edu/Tools/MPrime/ More Info: http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=mprime http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=mprime Note on its status: server unavailable since Dec. 2007.

22. 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

23. 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

24. 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

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

26. Public PCR Primers/Oligo Probes Repository – The NCBI Probe Database Web Site: http://www.ncbi.nlm.nih.gov/sites/entrez?db=probe 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

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

28. 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

29. 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

30. 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

31. Primer Design Tools for Multiplex PCR  MuPlex  PrimerStation

32. 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

33. 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

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

35. 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

36. 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

37. 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

38. 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

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

40. 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

41. 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

42. 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

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

44. 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

45. 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

46. 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

47. 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

48. 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

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. 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

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

55. 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

56. 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

57. 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

58. 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

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

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

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