Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany.
Nucleic Acids Res. 2010 Sep;38(16):e161. doi: 10.1093/nar/gkq572. Epub 2010 Jun 28.
Although the last few years have seen great progress in DNA sequence retrieval from fossil specimens, some of the characteristics of ancient DNA remain poorly understood. This is particularly true for blocking lesions, i.e. chemical alterations that cannot be bypassed by DNA polymerases and thus prevent amplification and subsequent sequencing of affected molecules. Some studies have concluded that the vast majority of ancient DNA molecules carry blocking lesions, suggesting that the removal, repair or bypass of blocking lesions might dramatically increase both the time depth and geographical range of specimens available for ancient DNA analysis. However, previous studies used very indirect detection methods that did not provide conclusive estimates on the frequency of blocking lesions in endogenous ancient DNA. We developed a new method, polymerase extension profiling (PEP), that directly reveals occurrences of polymerase stalling on DNA templates. By sequencing thousands of single primer extension products using PEP methodology, we have for the first time directly identified blocking lesions in ancient DNA on a single molecule level. Although we found clear evidence for blocking lesions in three out of four ancient samples, no more than 40% of the molecules were affected in any of the samples, indicating that such modifications are far less frequent in ancient DNA than previously thought.
尽管在过去的几年中，从化石标本中获取 DNA 序列方面取得了很大的进展，但有些古老 DNA 的特征仍然知之甚少。这在阻断性损伤方面尤其如此，即无法被 DNA 聚合酶绕过的化学改变，从而阻止受影响分子的扩增和随后的测序。一些研究得出结论，绝大多数古老的 DNA 分子都携带阻断性损伤，这表明，去除、修复或绕过阻断性损伤可能会极大地增加可供古 DNA 分析的标本的时间深度和地理范围。然而，之前的研究使用了非常间接的检测方法，这些方法并没有对内源性古老 DNA 中阻断性损伤的频率提供明确的估计。我们开发了一种新方法，即聚合酶延伸分析（PEP），它可以直接揭示聚合酶在 DNA 模板上的停顿情况。通过使用 PEP 方法对数千个单引物延伸产物进行测序，我们首次直接在单个分子水平上鉴定了古老 DNA 中的阻断性损伤。尽管我们在四个古老样本中的三个中发现了明确的阻断性损伤证据，但在任何一个样本中，只有不超过 40%的分子受到影响，这表明这种修饰在古老 DNA 中比之前认为的要罕见得多。
