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

在缺乏修复的情况下，损伤会在DNA中积累，最终破坏编码的遗传信息。因此，尽管DNA可能在古生物学、考古学或法医标本中存在相当长的时间，但它不可避免地会受到损害。然而，并不是所有的信息都不可挽回地丢失了。由于通常用于PCR扩增的DNA聚合酶在受损部位停滞不前，大部分DNA无法进入。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。宿主实验室已经证明，原则上，通常用于PCR和测序的聚合酶（如Taq DNA聚合酶）可以被赋予一种能力（尽管目前有限）绕过模板病变而不影响加工过程。该项目的目的是进一步设计和发展这种能力，并探索这种新型聚合酶的应用，以从史前动植物样本和考古标本中检索以前无法获取的DNA序列。

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.