Abstract
Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry’s disease (FD), steroid 21-hydroxylase deficiency (21-HD), and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations, which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases where other techniques have failed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Balogh, K., Patocs, A., Majnik, J., Racz, K., & Hunyady, L. (2004). Genetic screening methods for the detection of mutations responsible for multiple endocrine neoplasia type 1. Molecular Genetics and Metabolism, 83(1–2), 74–81.
Hofstra, R. M., Mulder, I. M., Vossen, R., de Koning-Gans, P. A., Kraak, M., Ginjaar, I. B., van der Hout, A. H., Bakker, E., Buys, C. H., van Ommen, G. J., van Essen, A. J., & den Dunnen, J. T. (2004). DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients. Human Mutation, 23(1), 57–66.
Buzin, C. H., Feng, J., Yan, J., Scaringe, W., Liu, Q., den Dunnen, J., Mendell, J. R., & Sommer, S. S. (2005). Mutation rates in the dystrophin gene: A hotspot of mutation at a CpG dinucleotide. Human Mutation, 25(2), 177–188.
Hamed, S. A., & Hoffman, E. P. (2006). Automated sequence screening of the entire dystrophin cDNA in Duchenne dystrophy: Point mutation detection. American Journal of Medical Genetics. Part B, Neuropsychiatric Genetics, 141(1), 44–50.
Davies, J. F., Redmond, E. K., Cox, M. C., Lalloo, F. I., Elles, R., & Evans, D. G. (2000). 2157delG: A frequent mutation in BRCA2 missed by PTT. Journal of Medical Genetics, 37, e42.
Shenk, T. E., Rhodes, C., Rigby, P. W., & Berg, P. (1975). Biochemical method for mapping mutational alterations in DNA with S1 nuclease: The location of deletions and temperature-sensitive mutations in simian virus 40. Proceedings of the National Academy of Sciences of the United States of America, 72(3), 989–993.
Yang, B., Wen, X., Kodali, N. S., Oleykowski, C. A., Miller, C. G., Kulinski, J., Besack, D., Yeung, J. A., Kowalski, D., & Yeung, A. T. (2000). Purification, cloning, and characterization of the CEL I nuclease. Biochemistry, 39(13), 3533–3541.
Shi, R., Otomo, K., Yamada, H., Tatsumi, T., & Sugawara, I. (2006). Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease. Microbes and Infection, 8(1), 128–135.
Till, B. J., Colbert, T., Codomo, C., Enns, L., Johnson, J., Reynolds, S. H., Henikoff, J. G., Greene, E. A., Steine, M. N., Comai, L., & Henikoff, S. (2006). High-throughput TILLING for Arabidopsis. Methods in Molecular Biology, 323, 127–135.
Wienholds, E., van Eeden, F. J., Kosters, M., Mudde, J., Plasterk, R. H., & Cuppen, E. (2003). Efficient target-selected mutagenesis in zebrafish. Genome Research, 13(2), 2700–2707.
Oleykowski, C. A., Bronson Mullins, C. R., Godwin, A. K., & Yeung, A. T. (1998). Mutation detection using a novel plant endonuclease. Nucleic Acids Research, 26(20), 4597–4602.
Yeung, A. T., Hattangadi, D., Blakesley, L., & Nicolas, E. (2005). Enzymatic mutation detection technologies. Biotechniques, 38(5), 749–758.
Shandilya, H., D’Alessio, J. M., O’Connor, K., Durocher, J., & Gerard, G. (2004). Mutation detection using Surveyor nuclease. Biotechniques, 36(4), 702–707.
Bannwarth, S., Procaccio, V., & Paquis-Flucklinger, V. (2005). Surveyor Nuclease: A new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects. Human Mutation, 25(6), 575–582.
Kanavakis, E., Efthymiadou, A., Strofalis, S., Doudounakis, S., Traeger-Synodinos, J., & Tzetis, M. (2003). Cystic fibrosis in Greece: Molecular diagnosis, haplotypes, prenatal diagnosis and carrier identification amongst high-risk individuals. Clinical Genetics, 63(5), 400–409.
Kekou, K., Florentin, L., & Metaxotou, C. (1998). 3′ Acceptor splice site mutation in intron 50 leads to mild Duchenne muscular dystrophy phenotype. Human Mutation, (Suppl 1), S209–S212.
Kekou, K., Mavrou, A., Florentin, L., Youroukos, S., Zafiriou, D. I., Skouteli, H. N., & Metaxotou, C. (1999). Screening for minor changes in the distal part of the human dystrophin gene in Greek DMD/BMD patients. European Journal of Human Genetics, 7(2), 179–187.
Madsen, K. M., Hasholt, L., Sorensen, S. A., Fermer, M. L., & Dahl, N. (1995). Two novel mutations (L32P) and (G85N) among five different missense mutations in six Danish families with Fabry’s disease. Human Mutation, 5(3), 277–278.
Wedell, A., & Luthman, H. (1993). Steroid 21-hydroxylase deficiency: Two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations. Human Molecular Genetics, 2(5), 499–504.
den Dunnen, J. T., & Paalman, M. H. (2003). Standardizing mutation nomenclature: Why bother? Human Mutation, 22(3), 181–182.
Till, B. J., Burtner, C., Comai, L., & Henikoff, S. (2004). Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Research, 32(8), 2632–2641.
Cotton, R. G., Rodrigues, N. R., & Campbell, R. D. (1988). Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proceedings of the National Academy of Sciences of the United States of America, 85(12), 4397–4401.
Qiu, P., Shandilya, H., & Gerard, G. F. (2005). A method for clone sequence confirmation using a mismatch-specific DNA endonuclease. Molecular Biotechnology, 29(1), 11–18.
Janne, P. A., Borras, A. M., Kuang, Y., Rogers, A. M., Joshi, V. A., Liyanage, H., Lindeman, N., Lee, J. C., Ηalmos, B., Maher, E. A., Distel, R. J., Meyerson, M., & Johnson, B. E. (2006). A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clinical Cancer Research, 12(3 Pt 1), 751–758.
Aartsma-Rus, A., van Deutekom, J. C., Fokkema, I. F., Van Ommen, G. J., & den Dunnen, J. T. (2006). Entries in the Leiden Duchenne muscular dystrophy mutation database: An overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve, 34(2), 135–144.
Acknowledgments
The authors would like to thank the Emeritus Prof. A. Metaxotou for the constant support and interest in the study of neuromuscular disorders. We would also like to thank Dr M Tzeti and M. Drakopoulou for their help as well as Miss K. Pouliou for providing controls with known mutations. This work was supported by Muscular Dystrophy Association in Greece (MDA HELLAS).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Vogiatzakis, N., Kekou, K., Sophocleous, C. et al. Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol. Mol Biotechnol 55, 1–9 (2013). https://doi.org/10.1007/s12033-007-0062-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-007-0062-9