Liu Chih Long, Bernstein Bradley E, Schreiber Stuart L
CSH Protoc. 2008 May 1;2008:pdb.top42. doi: 10.1101/pdb.top42.
INTRODUCTIONT7-based linear amplification of DNA (TLAD) was designed primarily to overcome the shortcomings of exponential amplification approaches. TLAD uses a linear amplification method based on the in vitro transcription (IVT) of template DNA by RNA polymerase from the T7 phage, a common strategy used in RNA amplification protocols. The TLAD protocol introduced here successfully addresses amplification fidelity issues encountered with use of random PCR (R-PCR) with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays). Thus, optimizations have been done with this particular application in mind. For other applications that require DNA instead of RNA to be the end point, reverse transcription is a necessary step that increases the cost and complexity of necessary molecular biological manipulations to the sample, when compared with PCR. However, IVT amplification does offer improved fidelity and a much higher maximum yield per single reaction. Thus, other techniques that require amplification of complex mixtures of randomly fragmented genomic DNA can also benefit from this method.
基于T7的DNA线性扩增（TLAD）主要是为克服指数扩增方法的缺点而设计的。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，这是RNA扩增方案中常用的策略。本文介绍的TLAD方案成功解决了在ChIP芯片方法中使用随机PCR（R-PCR）时遇到的扩增保真度问题（即从细胞裂解物的染色质免疫沉淀[ChIP]中回收的DNA用于随后在DNA微阵列上的分析）。因此，是考虑到这一特定应用进行了优化。对于其他需要以DNA而非RNA作为终点的应用，与PCR相比，逆转录是一个必要步骤，这增加了对样品进行必要分子生物学操作的成本和复杂性。然而，IVT扩增确实提供了更高的保真度和单次反应更高的最大产量。因此，其他需要扩增随机片段化基因组DNA复杂混合物的技术也可从该方法中受益。
