Abstract
There are a number of techniques available for the detection and characterization of mutations that take advantage of the flexibility and power of polymerase chain reaction (PCR) (Table 1). Sequencing remains the gold standard, but it is not very efficient as a screening tool, in particular when evaluating large numbers of samples, or multiple or extended stretches of DNA. f the available techniques, single-strand conformation polymorphism analysis (SSCP) appears as one of the most versatile techniques, amenable to automation, and optimal for screening for known or unknown mutations.
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References
Orita, M, Iwahana, H, Kanazawa, H., Hayashi, K., and Sekiya, T (1989) Detection of polymorphisms of human DNA by electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86, 2766–2770
Hayashi, K (1991) PCR-SSCP a rapid and sensitive method for detection of mutations in the genomic DNA PCR Meth Appl 1, 34–38
Iwahana, H., Yoshimoto, K, and Itakura, M (1992) Detection of point mutations by SSCP of PCR amplified DNA after endonuclease digestion BioTechniques 12, 64–66
Michaud, J., Brody, L C., and Steel, G (1992) Strand-separating conformational polymorphism analysis. efficacy of detection of point mutations in the human ornithine d-aminotransferase gene. Genomics 13, 389–394
Heym, B., Honoré, N, Truffot-Pernot, C., Banerjee, A., Schurra, C, Jacobs, W R, Jr., van Embden, J D A, Grosset, J H., and Cole, S T (1994) Implications of multidrug resistance for the future of short course chemotherapy of tuberculosis. a molecular study Lancet 344, 293–298
Telenti, A, Honoré, N., Bernasconi, C, March, J., Ortega, A., and Cole, S T. (1997) Genotypic assessment of isomazid and rifampin resistance in Mycobacterium tuberculosis. a blind study at reference laboratory level J Clin Microbiol 35, 719–723
Telenti, A., Imboden, P, Marchesi, F., Schmidheini, T, and Bodmer, T. (1993) Direct, automated detection of rifampm-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism Antimxrob Agents Chemother 37, 2054–2058.
Honoré, N and Cole, S T (1994) Streptomycin resistance in mycobacteria. Antimicrob Agents Chemother 38, 238–242
Cooksey, R. C, Morlock, G. P, McQueen, A, Glickman, S. E, and Crawford, J T. (1996) Characterization of streptomycin resistance mechanisms among Mycobacterium tuberculosis isolates from New York City Antimicrob Agents Chemother 40, 1186–1188.
Takiff, H E., Salazar, L., Guerrero, C., Philipp, W., Huang, W. M, Kreiswirth, B., Cole, S. T., Jacobs, W R, Jr, and Telenti, A. (1994) Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations Antimicrob Agents Chemother 38, 773–780
Scorpio, A and Zhang, Y (1996) Mutations in pncA, a gene encoding pyrazinamldase/nicotinamidase, cause resistance to the antituberculous drug pyrazmamide in tubercle bacillus. Nature Med 2, 662–667
Telenti, A., Philipp, W, Sreeratsan, S, Bernasconi, C., Stockbauer, K E, Wieles, B, Musser, J. M, Jacobs, W. R Jr (1997) The emb operon, a unique gene cluster of Mycobacterium, tuberculosis involved in resistance to ethanbutol. Nature Med 3, 567–570
Telenti, A. and Persing, D. H (1996) Novel strategies for the detection of drug resistance in Mycobacterium tuberculosis Res Microbiol. 147, 73–79
Peng, H., Du, M, Ji, J., Isaacson, P G., and Pan, L (1995) High resolution SSCP analysis using polyacrylamide agarose composite gel and a background-free silver staining method BioTechniques 19, 410–414.
Hongyo, T, Buzard, G S, Calve, R J, and Weghorst, C M (1993) “Cold SSCP.” a sample, rapid and non-radioactive method for optimized single strand conformation polymorphism analyses. Nucleic Acid Res 21, 3637–3642
Vector, T. C., Pretorius, G. S., Felix, J. V., Jordaan, A. M., and van Helden, P. D (1996) katG mutations in isoniazid-resistant strains of Mycobacterium tuberculosis are not infrequent. Antimicrob. Agents Chemother 40, 1572.
Kapur, V., Ling-Ling, L., Hamrick, M R., Plikaytis, B. B, Shinnick, T M, Telenti, A., Jacobs, W. R., Jr, Banerjee, A, Cole, S, Yuen, K. Y., Clarridge, J. E., III, Kreiswirth, B., and Musser, J. M. (1995) Rapid Mycobactermm species assignment and unambiguous identification of mutations associated with atimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencmg. Arch Pathol Lab. Med 119, 138–130.
Kwok, S., Kellog, D. E., and Spasic, D. (1990) Effects of primer-template mismatches on the polymerase chain reaction. HIV–l model studies. Nucleic Acid Res 18, 999–1005.
Uhl, J. R, Sandhu, G S., Kline, B C., and Cockerill, F.R (1996) PCR-RFLP detection of point mutations in the catalase-peroxidase gene (katG) of Mycobacterium tuberculosis associated with isoniazid resistance, in PCR Protocols for Emerging Infectious Diseases (Persing D. H, ed.), American Society for Microbiology, Washington, DC, pp. 144–149.
De Beenhouwer, H., Lhiang, Z., Jannes, G., Mijs, W., Machtelinck, L., Rossau, R., Traore, H., and Portaels, F. (1995) Rapid detection of rifampicm resistance in sputum and biopsy specimens from tuberculosis patients by PCR and line probe assay. Tubercle Lung Dis 76, 425–430.
Boerresen, A.-L., Hovig, E., and Smith-Soerensen, B., (1991) Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc. Natl. Acad. Set USA 88, 8405–8409.
Riesner, D., Steger, G, Wiese, U, Heibey, M, and Henco, K. (1992) Temperature-gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction. Electrophoresis 13, 632–636
Felmlee, T. A., Liu, Q., Whelen, C., Williams, D., Sommer, S. S, and Persing, D. H. (1995) Genotypic detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand conformation polymorphism and dideoxy fingerprinting. J Clin Microbiol 33, 1617–1623.
Williams, D. L, Waguespack, C, Eisenach, K., Crawford, J. T., Portaels, F., Salfinger, M., Nolan, C. N, Abe, C., Stich-Groh, V, and Gillis, T P. (1994) Characterization of rifampin resistance in pathogenic mycobacteria. Antimicrob. Agents Chemother 38, 2380–2386.
Lyamichev, V., Brow, M A D, and Dahlberg, J E. (1993) Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260, 778–783
Myers, R. M., Larm, Z, and Mamatts, T. (1985) Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA-DNA duplexes Science 230, 1242–1246.
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© 1998 Humana Press Inc., Totowa, NJ
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Telenti, A., Honoré, N., Cole, S.T. (1998). Detection of Mutations in Mycobacteria by PCR-SSCP (Single-Strand Conformation Polymorphism). In: Parish, T., Stoker, N.G. (eds) Mycobacteria Protocols. Methods in Molecular Biology™, vol 101. Humana Press. https://doi.org/10.1385/0-89603-471-2:423
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DOI: https://doi.org/10.1385/0-89603-471-2:423
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