Department of Cancer Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg-Illkirch, France.
Nat Protoc. 2012 Jan 26;7(2):328-38. doi: 10.1038/nprot.2011.447.
Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells. LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation coupled to massive parallel sequencing (ChIP-seq) has been achieved for transcription factors and epigenetic modification of chromatin histones with 1,000 to 5,000 cells. LinDA largely simplifies reChIP-seq experiments to monitor co-binding at chromatin target sites. The single-tube design of LinDA is ideal for handling ultrasmall amounts of DNA (<30 pg) and is compatible with automation. The actual hands-on working time is less than 6 h with one overnight reaction. The present protocol describes all materials and critical steps, and provides examples and controls for LinDA. Applications of LinDA for genome-wide analyses of biobank samples and for the study of chromatin conformation and nuclear architecture are in progress.
T7 聚合酶线性扩增 DNA（LinDA）是一种通用且强大的方法，可用于在细胞数量很少的情况下生成足够数量的 DNA，以进行全基因组研究。LinDA 可以与许多全局分析技术结合使用。事实上，已经可以使用 1000 到 5000 个细胞实现与大规模平行测序（ChIP-seq）相结合的转录因子和染色质组蛋白的表观遗传修饰的染色质免疫沉淀。LinDA 极大地简化了再 ChIP-seq 实验，以监测染色质靶位点的共结合。LinDA 的单管设计非常适合处理超少量的 DNA（<30pg），并且与自动化兼容。整个实验过程实际操作时间不到 6 小时，仅需过夜孵育 1 次。本方案描述了所有材料和关键步骤，并提供了 LinDA 的示例和对照。LinDA 已应用于生物库样本的全基因组分析以及染色质构象和核结构的研究。
