Efficient discovery of DNA polymorphisms in natural populations by Ecotilling
Efficient discovery of DNA polymorphisms in natural populations by Ecotilling
复制标题
DOI：
10.1111/j.0960-7412.2003.01999.x
复制
发表时间：
2004-03-01
期刊：
影响因子：
7.2
通讯作者：
Henikoff, S
中科院分区：
文献类型：
--
作者：
Comai, L;Young, K;Henikoff, S
我们采用了基因组靶向诱导局部病变（TILLING）中使用的突变检测技术，以发现自然人群中的多态性。将查询个体的基因组DNA与参考DNA混合，并用于用不对称标记的荧光引物扩增DNA的靶1-kbp区域。加热和退火后，异源双链体在错配位点被内切核酸酶CEL I切割，并使用Li-cor凝胶分析仪观察切割的链。在一个荧光通道中检测到的推定多态性可以通过在另一个通道中出现相反的切割链来验证。我们通过在150多个个体中发现5个基因的55种单倍型来证明这种称为Ecotilling的技术的效率，这些基因的序列从单核苷酸多态性不同的序列到代表复杂单倍型的序列。发现的多态性通过测序得到证实，包括碱基对变化、小的插入和缺失以及微卫星重复数的变化。Ecotilling允许快速检测许多个体中的变异，并且具有成本效益，因为每个单倍型仅需要测序一个个体。该技术适用于任何生物体，包括杂合和多倍体生物体。
We have adapted the mutation detection technology used in Targeting Induced Local Lesions in Genomes (TILLING) to the discovery of polymorphisms in natural populations. The genomic DNA of a queried individual is mixed with a reference DNA and used to amplify a target 1-kbp region of DNA with asymmetrically labeled fluorescent primers. After heating and annealing, heteroduplexes are nicked at mismatched sites by the endonuclease CEL I and cut strands are visualized using Li-cor gel analyzers. Putative polymorphisms detected in one fluorescence channel can be verified by appearance of the opposite cut strand in the other channel. We demonstrated the efficiency of this technology, called Ecotilling, by the discovery in 150+ individuals of 55 haplotypes in five genes, ranging from sequences differing by a single nucleotide polymorphism to those representing complex haplotypes. The discovered polymorphisms were confirmed by sequencing and included base-pair changes, small insertions and deletions, and variation in microsatellite repeat number. Ecotilling allows the rapid detection of variation in many individuals and is cost effective because only one individual for each haplotype needs to be sequenced. The technology is applicable to any organism including those that are heterozygous and polyploid.