School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India.
Plant Methods. 2010 Jan 26;6(1):3. doi: 10.1186/1746-4811-6-3.
TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetics procedure for identifying point mutations in selected gene(s) amplified from a mutagenized population using high-throughput detection platforms such as slab gel electrophoresis, capillary electrophoresis or dHPLC. One essential pre-requisite for TILLING is genomic DNA isolation from a large population for PCR amplification of selected target genes. It also requires multiplexing of genomic DNA isolated from different individuals (pooling) in typically 8-fold pools, for mutation scanning, and to minimize the number of PCR amplifications, which is a strenuous and long-drawn-out work. We describe here a simplified procedure of multiplexing, NEATTILL (Nucleic acid Extraction from Arrayed Tissue for TILLING), which is rapid and equally efficient in assisting mutation detection.
The NEATTILL procedure was evaluated for the tomato TILLING platform and was found to be simpler and more efficient than previously available methods. The procedure consisted of pooling tissue samples, instead of nucleic acid, from individual plants in 96-well plates, followed by DNA isolation from the arrayed samples by a novel protocol. The three variants of the NEATTILL procedure (vast, in-depth and intermediate) can be applied across various genomes depending upon the population size of the TILLING platform. The 2-D pooling ensures the precise confirmation of the coordinates of the positive mutant line while scanning complementary plates. Choice of tissue for arraying and nucleic acid isolation is discussed in detail with reference to tomato.
NEATTILL is a convenient procedure that can be applied to all organisms, the genomes of which have been mutagenized and are being scanned for multiple alleles of various genes by TILLING for understanding gene-to-phenotype relationships. It is a time-saving, less labour intensive and reasonably cost-effective method. Tissue arraying can cut costs by up to 90% and minimizes the risk of exposing the DNA to nucleases. Before arraying, different tissues should be evaluated for DNA quality, as the case study in tomato showed that cotyledons rather than leaves are better suited for DNA isolation. The protocol described here for nucleic acid isolation can be generally adapted for large-scale projects such as insertional mutagenesis, transgenic confirmation, mapping and fingerprinting which require isolation of DNA from large populations.
TILLING（靶向诱导基因组局部突变）是一种反向遗传学方法，用于从诱变群体中扩增的选定基因中鉴定点突变，使用高通量检测平台，如平板凝胶电泳、毛细管电泳或 dHPLC。TILLING 的一个基本前提是从大量群体中分离基因组 DNA，用于选定靶基因的 PCR 扩增。它还需要将来自不同个体的基因组 DNA（混合）在典型的 8 倍混合池中进行多重化，用于突变扫描，并最大限度地减少 PCR 扩增的数量，这是一项艰苦而漫长的工作。我们在这里描述了一种简化的多重化方法，NEATTILL（用于 TILLING 的阵列组织核酸提取），它快速且同样有效地辅助突变检测。
该 NEATTILL 程序已在番茄 TILLING 平台上进行了评估，结果发现该程序比以前的方法更简单、更高效。该程序包括在 96 孔板中混合个体植物的组织样本，而不是核酸，然后通过一种新的方案从排列样本中分离 DNA。该 NEATTILL 程序的三个变体（广泛、深入和中间）可以根据 TILLING 平台的群体大小应用于各种基因组。二维混合确保在扫描互补板时精确确认阳性突变系的坐标。参考番茄详细讨论了用于排列和核酸分离的组织选择。
NEATTILL 是一种方便的程序，可应用于所有已发生突变且正在通过 TILLING 扫描各种基因的多个等位基因以了解基因与表型关系的生物体。它是一种省时、劳动强度低且成本效益合理的方法。组织排列可以将成本降低 90%，并最大限度地减少 DNA 暴露于核酸酶的风险。在排列之前，应该评估不同的组织以获得 DNA 质量，正如番茄的案例研究表明，子叶比叶片更适合 DNA 分离。此处描述的用于核酸分离的方案通常可适用于大规模项目，例如插入诱变、转基因确认、图谱绘制和指纹图谱，这些项目需要从大量群体中分离 DNA。
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