Mechanism of chimera formation during the Multiple Displacement Amplification reaction.
Mechanism of chimera formation during the Multiple Displacement Amplification reaction.
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DOI：
10.1186/1472-6750-7-19
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发表时间：
2007-04-12
影响因子：
3.5
通讯作者：
Stockwell, Timothy B
中科院分区：
文献类型：
--
作者：
Lasken, Roger S;Stockwell, Timothy B
背景：多个位移放大（MDA）是一种用于扩增限制DNA源的方法。高分子量扩增的DNA是DNA文库结构的理想选择。尽管这已经使一个或几个无法培养的微生物细胞从一个或几个细胞中进行了基因组测序，但由于MDA在扩增的DNA中产生嵌合DNA重排的趋势使该过程变得复杂。确定DNA重排的来源将是迈向减少或消除它们的重要一步。星期：在这里，我们表征了通过从单个大肠杆菌细胞进行MDA整个基因组扩增而形成的主要类型的嵌合体，并通过454生命科学方法。对475个嵌合体的分析揭示了产生DNA重排的主要反应机制。在MDA中合成的高度分支DNA可以假设许多替代二级结构。在初始模板上延伸的DNA链可以位移，可以在创建嵌合体的第二个模板上使用。证据支持了一个模型，其中分支迁移可以使3'端释放到新模板上的质量。超过85％的DNA重排是模型预测的中间缺失的反向序列。分子内的重排受到青睐，并在同一分支DNA分子中包含的单链5'-strands移位3'末端。在70％以上的嵌合连接处，3'末端在新模板中以2-21个核苷酸（NTS）的免费序列开始启动启动。结论：嵌合体的形成是MDA方法的重要限制，特别是整个基因组方法测序。鉴定嵌合体形成机制为MDA反应提供了新的见解，并提出了减少嵌合体的方法。这里使用的454个测序方法将提供一种评估反应修饰效用的快速方法。
BACKGROUND: Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them.RESULTS: Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2-21 nucleotides (nts) in the new templates.CONCLUSION: Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications.