Engineering of polymerases for the amplification of damaged DNA: applications in paleobiology, archaeology and forensic medicine

用于扩增受损 DNA 的聚合酶工程：在古生物学、考古学和法医学中的应用

• 批准号: 35428683
• 负责人: Dr. Claudia Baar
• 金额: --
• 依托单位: Institut für Biochemie und Biologie
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2006
• 资助国家: 德国
• 起止时间: 2005-12-31 至 2008-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/35428683?language=en
• 关键词: Engineering; polymerases; amplification; damaged; DNA

In the absence of repair, lesions accumulate in DNA, eventually corrupting the encoded genetic information. Thus, although DNA may persist in specimens of paleontological, archaeological, or forensic interest for considerable periods of time, it is inevitably damaged. However, not all information is irretrievably lost. Much of it is simply inaccessible because the DNA polymerases commonly used for PCR amplification stall at the sites of damage. Here we propose to use protein engineering and directed evolution technologies to modify polymerases in such a way that they are able to bypass DNA lesions and amplify damaged DNA in PCR. The host laboratory has already shown that in principle the polymerases commonly used for PCR and sequencing (such as Taq DNA polymerase) can be endowed with an ability (although currently limited) to bypass template lesions without compromising processivity. The aim of the project is to further engineer and evolve this ability and explore the application of such novel polymerases for the retrieval of previously inaccessible DNA sequences from samples of prehistoric flora and fauna and from specimens of archaeological interest.

在没有修复的情况下，损伤在DNA中积累，最终破坏编码的遗传信息。因此，尽管DNA可能会在古生物学、考古学或法医学标本中保存相当长的时间，但它不可避免地会受到损坏。然而，并非所有信息都不可挽回地丢失。其中大部分是无法接近的，因为通常用于PCR扩增的DNA聚合酶在损伤部位停滞。在这里，我们建议使用蛋白质工程和定向进化技术来修饰聚合酶，使它们能够绕过DNA损伤并在PCR中扩增受损的DNA。宿主实验室已经表明，原则上，通常用于PCR和测序的聚合酶（如Taq DNA聚合酶）可以被赋予绕过模板病变而不损害持续合成能力的能力（尽管目前有限）。该项目的目的是进一步设计和发展这种能力，并探索此类新型聚合酶的应用，以从史前植物群和动物群样本以及具有考古意义的标本中检索以前无法获得的DNA序列。