Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor.
Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor.
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DOI：
10.1186/1471-2229-9-143
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发表时间：
2009-12-04
影响因子：
5.3
通讯作者：
Sharp P
中科院分区：
文献类型：
--
作者：
Dong C;Vincent K;Sharp P
耕作（基因组中靶向局部病变）是一种反向遗传学的功能，将传统的化学诱变与高通量PCR的突变检测结合在一起，发现诱导的突变，以改变蛋白质功能。使用核酸内切酶CELI的裂解测定。外显子中具有较高的家族的类似序列和介绍中的较低的家伙。或在缺乏方法的基因中，或者在这里没有有关方法的信息。使用突变测量师软件的混合PCR扩增子分析包含三个同源基因片段和序列分析的混合PCR扩增子，旨在简单地检测三个同源基因中的突变。
我们证明，高分辨率融化（HRM）分析可用于在包含三个同源基因片段的混合PCR放大中，使用突变测量师序列分析足以检测到序列分析的敏感性，足以检测在EMS中测试并验证了包含三个均种同性子的混合PCR扩增子（乙烷）硫酸盐耕种种群，淀粉合酶II（SSII）基因的羧基末端结构域中的筛查突变可以通过克隆来进一步分析，以确认突变并确定突变的基因组起源。
多倍体在基因的保守区域通常代表功能结构域，并且在同型基因座之间具有很高的序列相似性方法也可以用于多倍体物种中的SNP（单核丁基多态性）标记和生态灌输。
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. We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.
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实时甲基化特异性PCR（SMART-MSP）之后的敏感熔解分析：高通量和无探针定量DNA甲基化检测。
影响因子：
14.9
作者：
Kristensen, Lasse S.;Mikeska, Thomas;Krypuy, Michael;Dobrovic, Alexander
通讯作者：
Dobrovic, Alexander
影响因子：
9.3
作者：
Zhou, LM;Wang, L;Wittwer, CT
通讯作者：
Wittwer, CT
DOI：
10.1073/pnas.021506298
发表时间：
2001-01-16
影响因子：
11.1
作者：
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通讯作者：
Braun, A
影响因子：
3.8
作者：
Krypuy M;Newnham GM;Thomas DM;Conron M;Dobrovic A
通讯作者：
Dobrovic A
影响因子：
5.4
作者：
Wu, Shu-Biao;Wirthensohn, Michelle G.;Sedgley, Margaret
通讯作者：
Sedgley, Margaret