Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues
Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues
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DOI：
10.1186/1471-2164-7-312
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发表时间：
2006-12-12
期刊：
影响因子：
4.4
通讯作者：
Tsao, Ming Sound
中科院分区：
文献类型：
--
作者：
Aviel-Ronen, Sarit;Zhu, Chang Qi;Tsao, Ming Sound
背景：福尔马林固定石蜡包埋（FFPE）组织是可用于人类癌症基因组研究的档案生物材料的最大来源。因此，需要开发使用从 FFPE 组织中提取的 DNA 进行全基因组扩增 (WGA) 的方法。多链置换扩增 (MDA) 是一种使用 Bst DNA 聚合酶大片段的 WGA 等温方法。迄今为止，MDA 仅适用于从新鲜或速冻组织中分离的基因组 DNA，并且产生的代表性失真小于三倍。 结果：我们使用 Bst DNA 聚合酶的大片段扩增了正常人肺组织的五个 FFPE 样本的基因组 DNA。使用定量 PCR，评估了扩增 DNA 样本和原始 DNA 样本中 7 个基因的拷贝数。还评估了来自具有已知 N-myc 基因拷贝数的细胞系的 4 个神经母细胞瘤异种移植样本，以及具有已知 Skp2 基因扩增的 7 个非小细胞肺癌 (NSCLC) 肿瘤样本。此外，我们还比较了 Bst MDA 前后两个 NSCLC 样本基于阵列比较基因组杂交 (CGH) 的基因组图谱。 DNA 的中值扩增达到 990 倍。 DNA 扩增产物具有非常高的分子量 (> 23 Kb)。当扩增样本的基因内容与原始样本的基因内容进行比较时，代表性失真被限制在三倍。扩增和未扩增的 FFPE DNA 的阵列 CGH 基因组图谱相似。结论：大片段 Bst DNA 聚合酶适用于从 FFPE 组织中提取的 DNA 进行 WGA，预计最大代表性失真为三倍。扩增的 DNA 可用于通过定量实时 PCR 检测基因拷贝数变化，并通过阵列 CGH 进行基因组分析。
Background: Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA) is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold.Results: We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC) tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH)-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb). When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar.Conclusion: Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.