Pääbo S, Irwin D M, Wilson A C
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
J Biol Chem. 1990 Mar 15;265(8):4718-21.
Pairs of templates and primers were designed so that only recombination events would lead to amplification via the polymerase chain reaction. This approach reveals that lesions such as breaks, apurinic sites, and UV damage in a DNA template can cause the extending primer to jump to another template during the polymerase chain reaction. By comparing sequences of amplification products that were determined directly or via bacterial cloning, it was shown that when the thermostable Thermus aquaticus DNA polymerase encounters the end of a template molecule, it sometimes inserts an adenosine residue; the prematurely terminated product then jumps to another template and polymerization continues, creating an in vitro recombination product. Consequently, amplification products from damaged templates such as archaeological DNA are made up of a high proportion of chimeric molecules. The illegitimate adenosine and thymidine residues in these molecules are detected when cloned molecules are sequenced, but are generally averaged out when the amplification product is sequenced directly. However, if site-specific lesions exist in template DNA or if the amplification is initiated from very few copies, direct sequencing also may yield incorrect sequences. The phenomenon of the "jumping polymerase chain reaction" can be exploited to assess the frequency and location of lesions in nucleic acids.
设计了成对的模板和引物，以便只有重组事件会导致通过聚合酶链反应进行扩增。这种方法表明，DNA模板中的断裂、脱嘌呤位点和紫外线损伤等损伤会导致延伸引物在聚合酶链反应过程中跳到另一个模板上。通过比较直接测定或通过细菌克隆确定的扩增产物序列，发现当嗜热栖热菌DNA聚合酶遇到模板分子末端时，它有时会插入一个腺苷残基；然后过早终止的产物跳到另一个模板上，聚合继续进行，产生体外重组产物。因此，来自受损模板（如考古DNA）的扩增产物由高比例的嵌合分子组成。当对克隆分子进行测序时，可以检测到这些分子中不合法的腺苷和胸腺嘧啶残基，但当直接对扩增产物进行测序时，这些残基通常会被平均掉。然而，如果模板DNA中存在位点特异性损伤，或者扩增从极少的拷贝开始，直接测序也可能产生错误的序列。“跳跃聚合酶链反应”现象可用于评估核酸中损伤的频率和位置。
