Identifying Mutations by High Resolution Melting in a TILLING Population of Rice.

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ID: 58101
2019
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Abstract
Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.
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Authors Li, Shan;Yu, Ying-Ping;Liu, Song-Mei;Fu, Hao-Wei;Huang, Jian-Zhong;Shu, Qing-Yao;Tan, Yuan-Yuan;
Journal Journal of visualized experiments : JoVE
Year 2019
DOI
10.3791/59960
URL
Keywords
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