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Tytuł: Mutation detection by analysis of DNA heteroduplexes in TILLING populations of diploid species
Autor: Szurman-Zubrzycka, Miriam
Chmielewska, Beata
Gajewska, Patrycja
Szarejko, Iwona
Słowa kluczowe: Heteroduplex; CEL I endonuclease; High-resolution melting; TILLING
Data wydania: 2017
Źródło: J. Jankowicz-Cieslak, T. Tai, J. Kumlehn, B. Till (red.), “Biotechnologies for Plant Mutation Breeding” (S. 281-303). Cham : Springer
Abstrakt: In the beginning of mutation research, mutations could only be detected indirectly through the analysis of the phenotypic alterations that they caused. The detection of mutations at the DNA level became possible with the development of sequencing methods. Nowadays, there are many different methods and strategies that have been created for mutation detection, both in natural and mutagenised populations. The strategies differ in accuracy and sensitivity, as well as in the laboratory facilities, time, costs and efforts that are required. The majority of them involve the pooling of DNA samples and the amplification of a gene (fragment) of interest followed by heteroduplex formation. One of the popular strategies for mutation identification takes advantage of the specific endonuclease (e.g. CEL I) that recognises and cuts heteroduplexes precisely at the 3′ position of the mismatch site. The cleaved fragments are usually visualised through electrophoresis in a polyacrylamide gel using LI-COR sequencers, but agarose electrophoresis may also be used for this purpose, although with less sensitivity. A different mutation identification strategy, which is based on the high-resolution melting (HRM) technique, may be the method of choice when working with a short gene or a gene fragment whose length optimally does not exceed 400 bp.
DOI: 10.1007/978-3-319-45021-6_18
ISBN: 978-3-319-45019-3
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