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Introduction to Bioinformatics in Microbiology pp 81–102Cite as

Primer Design

Design of Oligonucleotide PCR Primers and Hybridization Probes

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Abstract

An overview of the application of oligonucleotide design is given in relation to applications involving exploratory investigations and diagnostics. The criteria for selection of oligonucleotides including lengths of PCR primers and products, lengths of oligonucleotide hybridization probes, and the principles for sequence comparison are introduced as well as rules for sequence-based prediction of amplification as well as prediction of Tm. Computer programs are suggested accounting for the different applications mainly related to PCR such as degenerate primers, multiplex PCR, nested PCRs and SNPs, and hybridization such as microarrays and in situ hybridization. The activity demonstrates the design of oligonucleotides for PCR amplification of single DNA sequences and primerBLAST for the design of diagnostic PCR primers.

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References

  • Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W.and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Ostell J, Pruitt KD, Sayers EW. 2018. GenBank. Nucleic Acids Res. 46(D1):D41–D47.

    Article  CAS  PubMed  Google Scholar 

  • Borer, P. N., Dengler, B., Tinoco, I. Jr. and Uhlenbeck, O. C. 1974. Stability of ribonucleic acid double-stranded helices. J. Mol. Biol. 15, 843–853

    Article  Google Scholar 

  • Breslauer, K. J., Frank, R., Blocker, H. and Marky, L. A. 1986. Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U. S. A. 83, 3746–3750.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Büssow, K., Hoffmann, S., Sievert, V. 2002. ORFer – retrieval of protein sequences and open reading frames from GenBank and storage into relational databases or text files. BMC Bioinformatics 23:40

    Article  Google Scholar 

  • Chen, S. H., Lin, C. Y., Cho, C. S., Lo, C. Z. and Hsiung, C. A. 2003. Primer Design Assistant (PDA): A web-based primer design tool. Nucleic Acids Res. 31, 3751–3754.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gadberry M. D., Malcomber S. T., Doust A. N., and Kellogg E. A. 2005. Primaclade--a flexible tool to find conserved PCR primers across multiple species. Bioinformatics 21:1263–1264.

    Article  CAS  PubMed  Google Scholar 

  • Dieffenbach, C. W., Lowe, T. M. J. and Dveksler, G. S. 1995. General concepts for PCR primer design. pp. 133–142. In PCR primer a laboratory manual. Cold Spring Harbor Lab. Press.

    Google Scholar 

  • Feng, S. and Tillier, E. R. 2007. A fast and flexible approach to oligonucleotide probe design for genomes and gene families. Bioinformatics 23, 1195–1202.

    Article  CAS  PubMed  Google Scholar 

  • Fredslund, J., Schauser, L., Madsen, L. H., Sandal, N. and Stougaard, J. 2005. PriFi: using a multiple alignment of related sequences to find primers for amplification of homologs. Nucleic Acids Res. 33, W516 - W520.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gibson, G. and Muse, S. V. 2004. A primer of genome science. Sinauer, Sunderland.

    Google Scholar 

  • Grunenwald, H. 2003. Optimization of polymerase chain reactions. In PCR Protocols. 2nd ed. ed. Bartlett, J. M. S. and Stirling, D. pp. 89–99. Methods in Molecular Biology 226. Humana Press, Totowa.

    Google Scholar 

  • Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95–98.

    CAS  Google Scholar 

  • Huggett, J. F., Foy, C. A., Benes, V., Emslie, K., Garson, J. A., Haynes, R., Hellemans, J., Kubista, M., Mueller, R. D., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., Wittwer, C. T., Bustin, S. A. 2013. The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. Clin Chem. 59, 892–902. doi: https://doi.org/10.1373/clinchem.2013.206375.

    Article  CAS  PubMed  Google Scholar 

  • Hyndman, D. L. and Mitsuhashi, M. 2003. PCR Primer Design. In PCR Protocols. 2nd ed. ed. Bartlett, J. M. S. and Stirling, D. pp. 81-88 Methods in Molecular Biology 226. Humana Press, Totowa.

    Google Scholar 

  • Ikuta, S., Takagi, K., Wallace, R. B., and Itakura, K. 1987. Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs. Nucleic Acids Res. 15, 797–811.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Karsch-Mizrachi I, Takagi T, Cochrane G; International Nucleotide Sequence Database Collaboration. 2018. The international nucleotide sequence database collaboration. Nucleic Acids Res. 46(D1):D48-D51.

    Google Scholar 

  • Ke, X., Collins, A. and Ye, S. 2001. PIRA PCR designer for restriction analysis of single nucleotide polymorphisms. Bioinformatics 17, 838–839.

    Article  CAS  PubMed  Google Scholar 

  • Kibbe, W. A. 2007. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res. 35, W43–6.

    Article  PubMed  PubMed Central  Google Scholar 

  • Kodama Y, Mashima J, Kosuge T, Kaminuma E, Ogasawara O, Okubo K, Nakamura Y, Takagi T. 2018. DNA Data Bank of Japan: 30th anniversary. Nucleic Acids Res. 46(D1):D30-D35.

    Article  CAS  PubMed  Google Scholar 

  • Kwok, S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C., and Sninsky, J. J. 1990. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 18, 999–1005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kwok, S., Chang, S-Y., Sninsky, J. J. and Wang, A. 1995. Desing and use of mismatched and degenerate primers. In Dieffenbach, C. W. and Dveksler, G. S. PCR primer a laboratory manual. pp. 143-155. Cold Spring Harbor Lab. Press.

    Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007): Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.

    Article  CAS  PubMed  Google Scholar 

  • Lathe, R. 1985. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J. Mol. Biol. 183, 1–12.

    Article  CAS  PubMed  Google Scholar 

  • Lawyer, F. C., Stoffel, S., Saiki, R. K., Myambo, K., Drummond, R., and Gelfand, D. H. 1989. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. J. Biol. Chem. 264, 6427–6437.

    CAS  PubMed  Google Scholar 

  • Li, F. and Stormo, G. D. 2001. Selection of optimal DNA oligos for gene expression arrays. Bioinformatics 17, 1067–76

    Article  CAS  PubMed  Google Scholar 

  • Ludwig, W., Strunk, O., Westram, R. and 29 other authors. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32, 1363–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McConaughy BL, Laird CD, McCarthy BJ. 1969. Nucleic acid reassociation in formamide. Biochemistry 8, 3289–3295.

    Article  CAS  PubMed  Google Scholar 

  • Meinkoth, J. and Wahl, G. 1984. Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138, 267–284.

    Article  CAS  PubMed  Google Scholar 

  • NCBI Resource Coordinators. 2016. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 44(D1):D7–19.

    Article  CAS  Google Scholar 

  • NCBI Resource Coordinators. 2018. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 46, D8-D13.

    Article  CAS  Google Scholar 

  • Newton, C. R., Graham, A., Heptinstall, L. E., Powell, S. J., Summers, C., Kalsheker, N., Smith, J. C. and Markham, A. F. (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17, 2503–2516.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nielsen, H. B. and Knudsen, S. 2002. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays. Bioinformatics 18, 321–322.

    Article  CAS  PubMed  Google Scholar 

  • Onodera, K. and Melcher, U. 2002. VirOligo: a database of virus-specific oligonucleotides. Nucleic Acids Res. 30, 203–204.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Owczarzy, R., Tataurov, A.V., Wu, Y., Manthey, J.A., McQuisten, K.A., Almabrazi, H.G., Pedersen, K.F., Lin, Y., Garretson, J., McEntaggart, N.O., Sailor, C.A., Dawson, R.B., Peek, A.S. 2008. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res. 36, W163–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pearson, W. R. and Lipman, D. J. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. U. S. A. 85, 2444–2448.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pozhitkov, A., Noble, P. A., Domazet-Loso, T., Nolte, A. W., Sonnenberg, R., Staehler, P., Beier, M. and Tautz, D. 2006. Tests of rRNA hybridization to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be predicted. Nucleic Acids Res. 34, e66.

    Article  PubMed  PubMed Central  Google Scholar 

  • Rachlin, J., Ding, C., Cantor, C. and Kasif, S. 2005. MuPlex: multi-objective multiplex PCR assay design. Nucleic Acids Res. 33, W544 - W547.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rice P, Longden I & Bleasby A. 2000. EMBOSS: The European Molecular Biology Open Software Suite. Trends Genetics 16:276–277.

    Article  CAS  Google Scholar 

  • Rose, T. M., Henikoff, J. G. and Henikoff, S. 2003. CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res. 31, 3763–3766.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rouillard, J. M., Zuker, M., and Gulari, E. 2003. OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Res. 31, 3057–3062.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rozen, S. and Skaletsky, H. 2000. Primer3 on the WWW for general users and for biologist programmers. pp. 365-386 In Misener, S. and Krawetz, S. A. Bioinformatics Methods and Protocols. Methods in Molecular Biology 132. Humana, Totowa.

    Google Scholar 

  • Rychlik, W. and Rhoads, R. E. 1989. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res. 17, 8543–8551.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rychlik, W., Spencer, W. J. and Rhoads, R. E. 1990. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 18, 6409–6412.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • SantaLucia, J. 1998. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Nath. Acad. Sci. U. S. A. 95, 1460–1465.

    Article  CAS  Google Scholar 

  • Schildkraut, C. 1965. Dependence of the melting temperature of DNA on salt concentration. Biopolymers 3, 195–208.

    Article  CAS  PubMed  Google Scholar 

  • Sharkey, F. H., Banat, I. M. and Marchant, R. 2004. Detection and quantification of gene expression in environmental bacteriology. Appl. Environ. Microbiol. 70, 3795–3806.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suggs, S. V., Hirose, T., Miyake, E. H., Kawashima, M. J., Johnson, K. I. and Wallace, R. B. 1981. In ICN-UCLA Symp. Dev. Biol. Using Purified Genes. Brown, D. D. (ed.) Acad. Press., New York. vol. 23, 683-693.

    Google Scholar 

  • Szostak, J. W., Stiles, J. I., Tye, B.-K., Chiu, P., Sherman, F. & Wu, R. 1979. Hybridization with synthetic oligonucleotides. Methods Enzymology 68, 419–428.

    Article  CAS  Google Scholar 

  • Tsai, M. F., Lin, Y. J., Cheng, Y. C., Lee, K. H., Huang, C. C., Chen, Y. T. and Yao, A. (2007) PrimerZ: streamlined primer design for promoters, exons and human SNPs, Nucleic Acids Res. 35, W63–W65).

    Article  PubMed  PubMed Central  Google Scholar 

  • Untergasser, A., Nijveen, H., Rao, X., Bisseling, T., Geurts, R. and Leunissen, J. A. M. (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. 35, W71 - W74.

    Article  PubMed  PubMed Central  Google Scholar 

  • Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B.C., Remm, M., Rozen, S.G. 2012. Primer3-new capabilities and interfaces. Nucleic Acids Res. 40:e115.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Urakawa, H., Noble, P. A., El Fantroussi, S., Kelly, J. J., Stahl, D. A. 2002. Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses. Appl. Environ. Microbiol. 68, 235–244.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weckx, S., De Rijk, P., Van Broeckhoven, C. and Del-Favero, J. 2004. SNPbox: web-based high-throughput primer design from gene to genome. Nucleic Acids Res. 32, W170–2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weckx, S., De Rijk, P., Van Broeckhoven, C. and Del-Favero, J. 2005. SNPbox: a modular software package for large-scale primer design. Bioinformatics 21, 385–7.

    Article  CAS  PubMed  Google Scholar 

  • Wong, M. L. and Medrano, J. F. 2005. Real-time PCR for mRNA quantitation. Biotechniques 39, 75–85.

    Article  CAS  PubMed  Google Scholar 

  • Xu, D., Li, G., Wu, L., Zhou, J. and Xu, Y. 2002. PRIMEGENS: robust and efficient design of gene-specific probes for microarray analysis. Bioinformatics 18, 1432–1437.

    Article  CAS  PubMed  Google Scholar 

Further Readings

  • Introduction to practical work with PCR as well to the historical background is found in Sambrook and Russell (2001).

    Google Scholar 

  • Sambrook and Russell. 2001. Molecular Cloning. A laboratory manual. CSHL Pres.

    Google Scholar 

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  1. Department of Veterinary Animal Sciences, University of Copenhagen, Copenhagen, Denmark

    Henrik Christensen & John Elmerdahl Olsen

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  1. Henrik Christensen
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  2. John Elmerdahl Olsen
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Correspondence to Henrik Christensen .

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  1. Department of Veterinary Animal Sciences, University of Copenhagen, Copenhagen, Denmark

    Henrik Christensen

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Christensen, H., Olsen, J.E. (2018). Primer Design. In: Christensen, H. (eds) Introduction to Bioinformatics in Microbiology. Learning Materials in Biosciences. Springer, Cham. https://doi.org/10.1007/978-3-319-99280-8_5

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