Comai Luca, Young Kim, Till Bradley J, Reynolds Steven H, Greene Elizabeth A, Codomo Christine A, Enns Linda C, Johnson Jessica E, Burtner Chris, Odden Anthony R, Henikoff Steven
Department of Biology, Box 355325, University of Washington, Seattle, WA 98195, USA.
Plant J. 2004 Mar;37(5):778-86. doi: 10.1111/j.0960-7412.2003.01999.x.
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.
我们已将用于基因组靶向诱导局部损伤（TILLING）的突变检测技术应用于自然群体中多态性的发现。将被检测个体的基因组DNA与参考DNA混合，并用不对称标记的荧光引物扩增1-kbp的目标DNA区域。加热和退火后，核酸内切酶CEL I在错配位点切开异源双链体，并用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多个个体中发现五个基因的55个单倍型证明了这项称为生态TILLING技术的效率，这些单倍型的序列差异从单个核苷酸多态性到代表复杂单倍型的序列不等。通过测序确认了发现的多态性，包括碱基对变化、小插入和缺失以及微卫星重复数的变异。生态TILLING允许快速检测许多个体中的变异，并且具有成本效益，因为每个单倍型只需要对一个个体进行测序。该技术适用于任何生物体，包括杂合和多倍体生物体。
