Development and validation of a T7 based linear amplification for genomic DNA.
Development and validation of a T7 based linear amplification for genomic DNA.
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DOI：
10.1186/1471-2164-4-19
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发表时间：
2003-05-09
期刊：
影响因子：
4.4
通讯作者：
Bernstein BE
中科院分区：
文献类型：
--
作者：
Liu CL;Schreiber SL;Bernstein BE
转录因子结合位点和组蛋白修饰模式的基因组图谱为基因调控网络和染色质结构的性质提供了独特的见解。这些系统性研究使用微阵列来分析通过染色质免疫沉淀分离的DNA的组成。为了获得足够用于微阵列分析的量，必须扩增分离的DNA。目前的方案使用基于PCR的方法以指数方式扩增。然而，指数扩增方案对偏差高度敏感。线性扩增策略使扩增偏倚最小化，并对mRNA表达分析产生了深远的影响。由于缺乏合适的标签如polyA尾，这些方案尚未应用于基因组DNA的分析。我们开发了一种新型的基因组DNA线性扩增方案。末端转移酶用于将聚T尾添加到DNA片段的末端。通过包括限制浓度的终止核苷酸ddCTP来确保尾长均匀性。使用T7-polyA引物衔接子的第二链合成产生适合于体外转录（IVT）的双链模板。使用这种方法，我们能够扩增低至2.5 ng的基因组DNA，同时保留起始材料的大小分布。相比之下，我们发现，PCR扩增偏向于更大的大小的物种。此外，广泛的基于微阵列的分析表明，我们的线性扩增方案比常用的基于PCR的方法更有效地保留了动态范围和物种代表性。我们提出了一种基于T7的基因组DNA线性扩增方案。验证研究和与现有方法的比较表明，纳入本协议将减少基因组作图实验中的扩增偏倚。
Genomic maps of transcription factor binding sites and histone modification patterns provide unique insight into the nature of gene regulatory networks and chromatin structure. These systematic studies use microarrays to analyze the composition of DNA isolated by chromatin immunoprecipitation. To obtain quantities sufficient for microarray analysis, the isolated DNA must be amplified. Current protocols use PCR-based approaches to amplify in exponential fashion. However, exponential amplification protocols are highly susceptible to bias. Linear amplification strategies minimize amplification bias and have had a profound impact on mRNA expression analysis. These protocols have yet to be applied to the analysis of genomic DNA due to the lack of a suitable tag such as the polyA tail. We have developed a novel linear amplification protocol for genomic DNA. Terminal transferase is used to add polyT tails to the ends of DNA fragments. Tail length uniformity is ensured by including a limiting concentration of the terminating nucleotide ddCTP. Second strand synthesis using a T7-polyA primer adapter yields double stranded templates suitable for in vitro transcription (IVT). Using this approach, we are able to amplify as little as 2.5 ng of genomic DNA, while retaining the size distribution of the starting material. In contrast, we find that PCR amplification is biased towards species of greater size. Furthermore, extensive microarray-based analyses reveal that our linear amplification protocol preserves dynamic range and species representation more effectively than a commonly used PCR-based approach. We present a T7-based linear amplification protocol for genomic DNA. Validation studies and comparisons with existing methods suggest that incorporation of this protocol will reduce amplification bias in genome mapping experiments.
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