A comprehensive assay for targeted multiplex amplification of human DNA sequences
A comprehensive assay for targeted multiplex amplification of human DNA sequences
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DOI：
10.1073/pnas.0803240105
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发表时间：
2008-07-08
影响因子：
11.1
通讯作者：
Davis, Ronald
中科院分区：
文献类型：
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作者：
Krishnakumar, Sujatha;Zheng, Jianbiao;Davis, Ronald
我们开发了一个强大的和可重复的方法来平行扩增人类序列，用于下游多重序列分析。我们称这种方法为SMART（间隔区多重扩增反应），它部分基于挂锁探针技术。作为原理的证明，我们使用SMART技术从人类基因组DNA中同时扩增485个范围从100到500 bp的人类外显子。在多次重复中，>90%的靶以高度均匀性成功扩增，70%的靶落在10倍范围内，并且所有产物的丰度都落在彼此的100倍范围内。我们使用长度>300个碱基的长挂锁探针（LPP）用于测定，并且这些探针的增加的长度允许捕获长度高达500 bp的人序列，这对于捕获大多数人外显子是最佳的。为了设计LPP，我们开发了一种方法，使用适当设计的dsDNA模板生成具有精确末端的ssDNA分子。所述模板具有工程化到其中的适当限制性位点，所述限制性位点可以被消化以产生适合于λ核酸外切酶消化的核苷酸突出端，从而从dsDNA产生单链探针。SMART技术是灵活的，可以很容易地适应在一个单一的反应多路复用成千上万的靶序列。
We developed a robust and reproducible methodology to amplify human sequences in parallel for use in downstream multiplexed sequence analyses. We call the methodology SMART (Spacer Multiplex Amplification Reaction), and it is based, in part, on padlock probe technology. As a proof of principle, we used SMART technology to simultaneously amplify 485 human exons ranging from 100 to 500 bp from human genomic DNA. In multiple repetitions, >90% of the targets were successfully amplified with a high degree of uniformity, with 70% of targets failing within a 10-fold range and all products falling within a 100-fold range of each other in abundance. We used long padlock probes (LPPs) >300 bases in length for the assay, and the increased length of these probes allowed for the capture of human sequences up to 500 bp in length, which is optimal for capturing most human exons. To engineer the LPPs, we developed a method that generates ssDNA molecules with precise ends, using an appropriately designed dsDNA template. The template has appropriate restriction sites engineered into it that can be digested to generate nucleotide overhangs that are suitable for lambda exonuclease digestion, producing a single-stranded probe from dsDNA. The SMART technology is flexible and can be easily adapted to multiplex tens of thousands of target sequences in a single reaction.