Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor? |
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Authors: | Chongmei Dong Kate Vincent and Peter Sharp |
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Institution: | (1) Plant Breeding Institute, University of Sydney, PMB 4011, Narellan, NSW, 2567, Australia;(2) Australian Centre for Plant Functional Genomics, PMB 1, Glen Osmond, SA, 5064, Australia |
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Abstract: | Background TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical
mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The
most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method,
locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low
homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design
locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking
introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution
Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation
Surveyor? software, aimed at simultaneous detection of mutations in three homoeologous genes. |
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