DNA amplification method tolerant to sample degradation
DNA amplification method tolerant to sample degradation
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DOI：
10.1101/gr.2813404
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发表时间：
2004-11-01
期刊：
影响因子：
7
通讯作者：
Makrigiorgos, GM
中科院分区：
文献类型：
--
作者：
Wang, G;Maher, E;Makrigiorgos, GM
尽管最近在完整DNA/RNA的线性全基因组扩增方面取得了进展，但以完全无偏的方式扩增降解的核酸仍然是遗传诊断的一个严重挑战。我们描述了一种新的全基因组扩增程序，RCA-RCA(限制和环化辅助的滚环扩增)，它保留了退化的扩增基因组之间的等位基因差异，同时实现了几乎完全的基因组覆盖。RCA-RCA利用限制性内切酶消化和全基因组环化来产生适合滚环扩增的基因组序列。当使用完整的基因组DNA时，RCA-RCA在全基因组范围内保留了复杂基因组之间的基因扩增差异(两倍或更高)，与包括多重置换扩增在内的其他扩增方法相比，提供了与未扩增材料高度一致的一致性。使用RCA-RCA，福尔马林固定的DNA轻度或实质性降解的样本被成功地扩增，并通过阵列-CGH或Taqman PCR进行筛选，显示保留了原始材料的主要基因扩增特征。微卫星分析表明，RCA-RCA扩增的基因组DNA在核苷酸水平上代表了原始材料。通过RCA-RCA成功地扩增了基因，并进行了无偏基因表达分析(R2=0.99)。RCA-RCA的简单性和普适性使其成为一种强大的新的基因组分析工具，与以往的扩增技术相比具有独特的优势。
Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in all unbiased fashion remains a serious challenge for genetic diagnosis. We describe a new whole genome amplification procedure, RCA-RCA (Restriction and Circularization-Aided Rolling Circle Amplification), which retains the allelic differences among degraded amplified genomes while achieving almost complete genome coverage. RCA-RCA utilizes restriction digestion and whole genome circularization to generate genomic sequences amenable to rolling circle amplification. When intact genomic DNA is used, RCA-RCA retains gene-amplification differences (twofold or higher) between complex genomes on a genome-wide scale providing highly improved concordance with unamplified material as compared with other amplification methodologies including multiple displacement amplification. Using RCA-RCA, formalin-fixed samples of modest or substantial DNA degradation were successfully amplified and screened via array-CGH or Taqman PCR that displayed retention of the principal gene amplification features of the original material. Microsatellite analysis revealed that RCA-RCA amplified genomic DNA is representative of the original material at the nucleotide level. Amplification of cDNA is successfully performed via RCA-RCA and results to unbiased gene expression analysis (R-2 = 0.99). The simplicity and universal applicability of RCA-RCA make it a powerful new tool for genome analysis With unique advantages over previous amplification technologies.