Effect of highly fragmented DNA on PCR
Effect of highly fragmented DNA on PCR
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DOI：
10.1093/nar/24.24.5026
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发表时间：
1996-12-15
影响因子：
14.9
通讯作者：
Weihs, P
中科院分区：
文献类型：
--
作者：
Golenberg, EM;Bickel, A;Weihs, P
本研究以降解的DNA为模板，对聚合酶链式反应（PCR）的反应行为进行了研究，首先证明了在常规PCR之前，如果允许重叠片段退火和延伸，则可以扩增出大于初始模板片段的片段，当降解的基因组DNA在没有特异性引物的情况下通过聚合预处理时，扩增产物增加。我们测量了作为模板DNA降解的函数的核苷酸摄取，dNTP掺入最初随着DNA片段化的增加而增加，然后当DNA高度降解时下降，我们证明，dNTP摄取持续> 10个聚合循环，并受模板DNA的质量和数量以及底物dNTP的量的影响。这些结果表明，尽管降解DNA的重建可能允许扩增大片段，由于Tag聚合酶的末端脱氧核苷酸转移酶活性可能抑制3'退火并限制模板重建的长度，因此我们提出了将重建聚合反应和扩增聚合反应分开的改良PCR技术。
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 Tag polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.