Kilo-base Range Isothermal DNA Amplification

千碱基范围等温 DNA 扩增

• 批准号: 6934395
• 负责人: Huimin Kong
• 金额: $ 37.48万
• 依托单位: BIOHELIX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6934395
• 关键词: DNA directed DNA polymerase; enzyme activity; exodeoxyribonuclease; helicase; nucleic acid amplification techniques; plasmids; technology /technique development

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.

描述(由申请人提供)：扩增并随后检测有限长度的目标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，如克隆基因和分析遗传变异，往往需要有选择地扩增千碱基范围内的大DNA片段。目前，聚合酶链式反应(PCR)是研究人员唯一用来扩增大片段DNA的方法。尽管聚合酶链式反应能够扩增出10-20kb的靶标，但由于Taq聚合酶缺乏校对活性以及需要热循环，相对较高的错误率可能会限制聚合酶链式反应的使用。该研究提供了一种在恒温下指数放大中程和长程DMA片段的方法。最近发展了一种新的等温DNA扩增方法。这种扩增技术称为解旋酶依赖的扩增(HDA)，它基于DNA解旋酶的解离活性来分离两条DNA链，生成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自然界复制基因组DNA的选择。因此，这可能是一种更好的扩增DNA的方法。在第一阶段的研究中，我们证明了利用高进行性解旋酶RecBCD可以扩增出1kb的DNA片段，并且通过解旋酶和DNA聚合酶的协同作用来增加扩增片段的大小是可行的。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，扩增长DNA片段只需将DNA样本与HDA试剂混合，并在水浴中孵化一小时即可。