Liu Chih Long, Bernstein Bradley E, Schreiber Stuart L
CSH Protoc. 2008 May 1;2008:pdb.prot5004. doi: 10.1101/pdb.prot5004.
INTRODUCTIONT7-based linear amplification of DNA (TLAD) uses a linear amplification approach based on in vitro transcription (IVT) of template DNA by RNA polymerase from the T7 phage. TLAD was designed primarily for use with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays) and requires nanogram quantities of dsDNA to generate microgram amounts of amplified RNA. Briefly, the strategy is to add a 3' conserved end to the template dsDNA, using terminal deoxynucleotidyl transferase (TdT) tailing, which permits the addition of a T7 promoter sequence in the subsequent second-strand synthesis step. IVT can then use this newly appended T7 promoter and linearly amplify the template dsDNA, producing antisense RNA (aRNA) product. After the IVT reaction is complete, the aRNA is cleaned up using the QIAGEN RNeasy Kit. This protocol for RNA sample purification is based on the manufacturer's protocol for cleaning up RNA reactions, with minor modifications.
引言
基于T7的DNA线性扩增（TLAD）采用一种基于T7噬菌体RNA聚合酶对模板DNA进行体外转录（IVT）的线性扩增方法。TLAD主要设计用于与芯片免疫沉淀法（ChIP-chip）配合使用（即从细胞裂解物的染色质免疫沉淀（ChIP）中回收的DNA用于后续在DNA微阵列上的分析），并且需要纳克级量的双链DNA（dsDNA）来生成微克级量的扩增RNA。简而言之，该策略是使用末端脱氧核苷酸转移酶（TdT）加尾为模板dsDNA添加一个3'保守末端，这允许在随后的第二链合成步骤中添加T7启动子序列。然后，IVT可以使用这个新添加的T7启动子并线性扩增模板dsDNA，产生反义RNA（aRNA）产物。IVT反应完成后，使用QIAGEN RNeasy试剂盒纯化aRNA。该RNA样品纯化方案基于制造商用于纯化RNA反应的方案，并做了一些小的修改。
