Summary
Whole-genome polymerase chain reaction (PCR) scanning (WGPS) is based on the PCR amplification of small-sized chromosomes (e.g., bacterial chromosomes) by long-range PCR with a set of primers designed using a reference strain and applied to amplify several other strains. Such an approach of genome variability has specific requirements for the selection of primers and the design of primer pairs for the optimal coverage of the chromosome. To facilitate such analysis, we have developed GenoFrag, a software package for the design of primers optimized for whole-genome scanning by long-range PCR. GenoFrag works in a two-step procedure: first, a list of primers is selected according to the basic criteria, and second, the list of primer candidates is used for the coverage of the whole chromosome. These two steps are presented here with a part of the algorithm scripts developed for this software. Examples of what can be done using GenoFrag are illustrated by results obtained from the online version of the software. GenoFrag has already been validated in long-range (LR)-PCR experiment on several bacterial species. It is a robust and reliable tool for primer design for WGPS.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ohnishi, M., Terajima, J., Kurokawa, K., Nakayama, K., Murata, T., Tamura, K., Ogura, Y., Watanabe, H., and Hayashi, T. (2002) Proc Natl Acad Sci USA 99, 17043–8.
Lapidus, A., Galleron, N., Andersen, J. T., Jorgensen, P. L., Ehrlich, S. D., and Sorokin, A. (2002) FEMS Microbiol Lett 209, 23–30.
Beres, S. B., Sylva, G. L., Sturdevant, D. E., Granville, C. N., Liu, M., Ricklefs, S. M., Whitney, A. R., Parkins, L. D., Hoe, N. P., Adams, G. J., Low, D. E., DeLeo, F. R., McGeer, A., and Musser, J. M. (2004) Proc Natl Acad Sci USA 101, 11833–8.
Ben Zakour, N., Gautier, M., Andonov, R., Lavenier, D., Cochet, M. F., Veber, P., Sorokin, A., and Le Loir, Y. (2004) Nucleic Acids Res 32, 17–24.
Ben Zakour, N., Grimaldi, C., Gautier, M., Langella, P., Azevedo, V., Maguin, E., and Le Loir, Y. (2005) Res Microbiol 157, 386–94.
Rozen, S., and Skaletsky, H. (2000) Methods Mol Biol 132, 365–86.
Haas, S., Vingron, M., Poustka, A., and Wiemann, S. (1998) Nucleic Acids Res 26, 3006–12.
Li, P., Kupfer, K. C., Davies, C. J., Burbee, D., Evans, G. A., and Garner, H. R. (1997) Genomics 40, 476–85.
Gorelenkov, V., Antipov, A., Lejnine, S., Daraselia, N., and Yuryev, A. (2001) Biotechniques 31, 1326–30.
Rychlik, W. (1995) Mol Biotechnol 3, 129–34.
Suggs, S. V., Hirose, T., Myoke, E. H., Kawashima, M. J., Johnson, K. I., and Wallace, R. B. (1981) In Brown, D. D. (ed.), ICN-UCLA Symposium for Developmental Biology Using Purified Gene. Academic press, New York, 683–93.
Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A. (1986) Proc Natl Acad Sci USA 83, 3746–50.
Blommers, M. J., Walters, J. A., Haasnoot, C. A., Aelen, J. M., van der Marel, G. A., van Boom, J. H., and Hilbers, C. W. (1989) Biochemistry 28, 7491–8.
Kernighan, B. W., and Ritchie, D. M. (1988) The C Programming Language, Prentice Hall Professional Technical Reference.
Stajich, J. E., Block, D., Boulez, K., Brenner, S. E., Chervitz, S. A., Dagdigian, C., Fuellen, G., Gilbert, J. G. R., Korf, I., Lapp, H., Lehvaslaiho, H., Matsalla, C., Mungall, C. J., Osborne, B. I., Pocock, M. R., Schattner, P., Senger, M., Stein, L. D., Stupka, E., Wilkinson, M. D., and Birney, E. (2002) Genome Res 12, 1611–8.
Kurtz, S., Choudhuri, J. V., Ohlebusch, E., Schleiermacher, C., Stoye, J., and Giegerich, R. (2001) Nucleic Acids Res 29, 4633–42.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J Mol Biol 215, 403–10.
Kreiswirth, B. N., Lutwick, S. M., Chapnick, E. K., Gradon, J. D., Lutwick, L. I., Sepkowitz, D. V., Eisner, W., and Levi, M. H. (1995) Microb Drug Resist 1, 307–13.
Schneider, D., Duperchy, E., Depeyrot, J., Coursange, E., Lenski, R., and Blot, M. (2002) BMC Microbiol 2, 18.
Yarwood, J. M., McCormick, J. K., Paustian, M. L., Orwin, P. M., Kapur, V., and Schlievert, P. M. (2002) J Biol Chem 277, 13138–47.
Ubeda, C., Tormo, M. A., Cucarella, C., Trotonda, P., Foster, T. J., Lasa, I., and Penades, J. R. (2003) Mol Microbiol 49, 193–210.
Andonov, R., Lavenier, D., Veber, P., and Yanev, N. (2005) Concurr Comput: Pract Exper 17, 1657–68.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press
About this protocol
Cite this protocol
Zakour, N.B., Le Loir, Y. (2007). Designing Primers for Whole Genome PCR Scanning Using the Software Package GenoFrag. In: Yuryev, A. (eds) PCR Primer Design. Methods in Molecular Biology™, vol 402. Humana Press. https://doi.org/10.1007/978-1-59745-528-2_18
Download citation
DOI: https://doi.org/10.1007/978-1-59745-528-2_18
Publisher Name: Humana Press
Print ISBN: 978-1-58829-725-9
Online ISBN: 978-1-59745-528-2
eBook Packages: Springer Protocols