Kilo-base Range Isothermal DNA Amplification

描述（由申请人提供）：能够扩增和随后检测有限长度的目标序列，可能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基碱基对范围内的大片段DNA。聚合酶链反应（PCR）是目前研究人员扩增大片段DNA的唯一方法。虽然PCR能够扩增到10-20 kb的目标，但由于缺乏Taq聚合酶的校对活性和热循环的要求，相对较高的错误率可能限制了PCR的使用。本研究提出了一种在恒温条件下指数扩增中长距离DMA片段的方法。最近发展了一种新的等温DNA扩增方法。这种扩增技术，解旋酶依赖扩增（HDA），是基于DNA解旋酶的解绕活性来分离两条DNA链，产生单链模板，用于引物杂交和随后的DNA聚合酶延伸。这种扩增方法更接近于基因组DNA复制的自然选择。因此，这可能是一种更好的扩增DNA的方法。在I期研究中，我们证明了利用高进程解旋酶RecBCD可以扩增1 kb的DNA片段，并且可以通过解旋酶和DNA聚合酶的协同作用来增加扩增子的大小。因此，我们建议进一步优化基于recbcd的HDA体系，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。最终将开发的拟议产品将是基于recbcd的核酸扩增试剂盒，能够在一个温度下以高保真度进行千碱基范围的扩增。因此，长DNA片段的扩增只需将DNA样品与HDA试剂混合并在水浴中孵育一小时即可。

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.