d'Abbadie Marc, Hofreiter Michael, Vaisman Alexandra, Loakes David, Gasparutto Didier, Cadet Jean, Woodgate Roger, Pääbo Svante, Holliger Philipp
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Nat Biotechnol. 2007 Aug;25(8):939-43. doi: 10.1038/nbt1321. Epub 2007 Jul 15.
In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.
在缺乏修复的情况下，DNA损伤会不断累积。因此，保存在古生物学、考古学或法医学样本中的DNA不可避免地会受到损伤。我们描述了一种从受损DNA中恢复遗传信息的策略。通过对嗜热栖热菌属（嗜热水生栖热菌Taq、嗜热栖热菌Tth和嗜热栖热菌Tfl）的聚合酶基因进行分子育种以及区室化自我复制筛选，我们进化出了能够延伸单错配、双错配甚至四重错配、处理非经典引物-模板双链体并绕过古代DNA中发现的损伤（如乙内酰脲和无碱基位点）的聚合酶。将这些聚合酶应用于47000至60000年前洞熊DNA的PCR扩增时，其性能比Taq DNA聚合酶高出150%，并且在Taq无法扩增的样本稀释度下仍能产生扩增产物。我们的结果表明，工程化聚合酶可以扩大从更新世样本中恢复遗传信息的范围，可能有益于古生物学、考古学和法医学中的遗传分析。
