Ramification amplification: A novel isothermal DNA amplification method
Ramification amplification: A novel isothermal DNA amplification method
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DOI：
10.2165/00066982-200106020-00010
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发表时间：
2001-06-01
期刊：
影响因子：
--
通讯作者：
Li, HB
中科院分区：
文献类型：
--
作者：
Zhang, DY;Brandwein, M;Li, HB
我们开发了一种新的等温DNA扩增方法，其扩增机制与常规PCR完全不同。该方法使用特别设计的环状探针（C-探针），其中3 '和5 '末端通过与靶标杂交而并置在一起。然后通过T4 DNA连接酶以靶依赖性方式共价连接两个末端，产生闭合的DNA环。在存在过量引物（正向和反向引物）的情况下，DNA聚合酶使结合的正向引物沿着C探针延伸并置换下游链，产生多聚体单链DNA（ssDNA），类似于体内噬菌体的“滚环”复制。然后，该多聚体ssDNA用作多个反向引物的模板，以杂交、延伸和置换下游DNA，产生大的分枝（分支）DNA复合物。这个分支过程持续到所有ssDNA变成双链，导致指数扩增，其将自身与先前描述的非指数滚环扩增区分开。在这份报告中，我们证明了分支放大的原则。通过使用独特的噬菌体DNA聚合酶，空集29 DNA聚合酶，具有内在的高持续合成能力，我们能够在35 ℃下1小时内实现显着扩增。此外，我们应用此技术原位检测Raji细胞中的EB病毒序列。
We have developed a novel isothermal DNA amplification method with an amplification mechanism quite different from conventional PCR. This method uses a specially designed circular probe (C-probe) in which the 3 ' and 5 ' ends are brought together in juxtaposition by hybridization to a target. The two ends are then covalently linked by a T4 DNA ligase in a target-dependent manner, producing a closed DNA circle. In the presence of an excess of primers (forward and reverse primers), a DNA polymerase extends the bound forward primer along the C-probe and displaces the downstream strand, generating a multimeric single-stranded DNA (ssDNA), analogous to the "rolling circle" replication of bacteriophages in vivo. This multimeric ssDNA then serves as a template for multiple reverse primers to hybridize, extend, and displace downstream DNA, generating a large ramified (branching) DNA complex. This ramification process continues until all ssDNAs become double-stranded, resulting in an exponential amplification that distinguishes itself from the previously described nonexponential rolling circle amplification. In this report, we prove the principle of ramification amplification. By using a unique bacteriophage DNA polymerase, empty-set29 DNA Polymerase, that has an intrinsic high processivity, we are able to achieve significant amplification within 1 hour at 35 degreesC. In addition, we applied this technique for in situ detection of Epstein-Barr viral sequences in Raji cells.