Kilo-base Range Isothermal DNA Amplification

描述（由申请人提供）：扩增和随后检测具有有限长度的靶序列的能力可以满足诊断某些疾病的需要。然而，许多生物医学研究，如克隆基因和遗传变异分析，往往需要选择性扩增的大DNA片段在千碱基对范围。目前，聚合酶链式反应（PCR）是研究人员用来扩增大DNA片段的唯一方法。虽然PCR能够扩增高达10-20 kb的靶，但由于缺乏Taq聚合酶的校对活性和热循环的要求而导致的相对高的错误率可能限制PCR的使用。该研究为在恒温条件下以指数方式扩增中、远程DNA片段提供了一种新的方法。最近发展了一种新的等温DNA扩增方法。这种扩增技术，解旋酶依赖性扩增（HDA），是基于DNA解旋酶的解旋活性，以分离两条DNA链，产生单链模板，用于引物杂交和随后通过DNA聚合酶的延伸。这种扩增方法更接近自然选择的基因组DNA复制。因此，它可能是扩增DNA的更好方法。在第一阶段的研究中，我们证明，通过使用高度进行性的解旋酶RecBCD，我们可以扩增1 kb的DNA片段，并且通过解旋酶和DNA聚合酶的协调活性来增加扩增子的大小是可行的。因此，我们建议进一步优化基于RecBCD的HDA系统，并通过调节解旋酶和DNA聚合酶的活性来提高HDA反应的协调性。最终开发的拟议产品将是一种基于RecBCD的核酸扩增试剂盒，能够在一个温度下高保真地进行千碱基范围的扩增。因此，长DNA片段的扩增可以仅涉及将DNA样品与HDA试剂混合物混合并在水浴中孵育1小时。

DESCRIPTION (provided by applicant): The ability to amplify and subsequent detection a target sequence with a limited length may satisfy the need for diagnosing some of the diseases. Nevertheless, much biomedical research, such as cloning genes and analysis of genetics variation, often demands selective amplification of large DNA fragments in the kilo basepairs range. Currently Polymerase Chain Reaction (PCR) is the only method used by researchers to amplify large DNA fragments. Although PCR is able to amplify a target up to 10-20 kb, a relatively high error rate due to the lack of proof-reading activity of Taq polymerase and the requirement of thermo-cycling may limit the use of PCR. The research proposed provides an alternative method to exponentially amplify medium range and long range DMA fragment at a constant temperature. A new isothermal DNA amplification method has been recently developed. This amplification technology, Helicase-Dependent Amplification (HDA), is based on the unwinding activity of a DNA helicase to separate two DNA strands generating single-stranded templates for primer hybridization and subsequent extension by a DNA polymerase. This amplification method is closer to nature's choice for the replication of genomic DNA. Thus, it may be a better way to amplify DNA. During the Phase I research, we demonstrated that, by using highly processive helicase RecBCD, we could amplify 1 kb DNA fragment and that it is feasible to increase the size of amplicon by coordinate activities of helicase and DNA polymerase. Therefore, we propose to further optimize the RecBCD-based HDA system and to improve coordination of HDA reaction by modulating the activity of helicase and DNA polymerase. The proposed product ultimately to be developed will be a RecBCD-based nucleic acid amplification kit capable of doing kilo-base range amplification at one temperature with high fidelity. Thus, amplification of a long DNA fragment could merely involve mixing a DNA sample with the HDA reagent mix and incubation in a water bath for one hour.