Golenberg E M, Bickel A, Weihs P
Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA.
Nucleic Acids Res. 1996 Dec 15;24(24):5026-33. doi: 10.1093/nar/24.24.5026.
We characterized the behavior of polymerase chain reactions (PCR) using degraded DNA as a template. We first demonstrated that fragments larger than the initial template fragments can be amplified if overlapping fragments are allowed to anneal and extend prior to routine PCR. Amplification products increase when degraded genomic DNA is pretreated by polymerization in the absence of specific primers. Secondly, we measured nucleotide uptake as a function of template DNA degradation. dNTP incorporation initially increases with increasing DNA fragmentation and then declines when the DNA becomes highly degraded. We demonstrated that dNTP uptake continues for >10 polymerization cycles and is affected by the quality and quantity of template DNA and by the amount of substrate dNTP. These results suggest that although reconstruction of degraded DNA may allow amplification of large fragments, reconstructive polymerization and amplification polymerization may compete. This was confirmed in PCR where the addition of degraded DNA reduced the resultant product. Because terminal deoxynucleotidyl transferase activity of Taq polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.
我们对以降解DNA为模板的聚合酶链反应（PCR）行为进行了表征。我们首先证明，如果在常规PCR之前允许重叠片段退火并延伸，那么大于初始模板片段的片段可以被扩增。当在没有特异性引物的情况下通过聚合作用对降解的基因组DNA进行预处理时，扩增产物会增加。其次，我们测量了核苷酸摄取量与模板DNA降解程度的函数关系。dNTP掺入最初随着DNA片段化程度的增加而增加，然后在DNA高度降解时下降。我们证明dNTP摄取在超过10个聚合循环中持续存在，并且受模板DNA的质量和数量以及底物dNTP量的影响。这些结果表明，尽管降解DNA的重建可能允许大片段的扩增，但重建性聚合和扩增性聚合可能会相互竞争。这在PCR中得到了证实，即添加降解DNA会减少所得产物。由于Taq聚合酶的末端脱氧核苷酸转移酶活性可能会抑制3'端退火并限制模板重建的长度，我们建议采用改进的PCR技术来分离重建性聚合反应和扩增性聚合反应。
