Harris S, Jones D B
University Pathology, Southampton University NHS Trust, England, UK.
Br J Biomed Sci. 1997 Sep;54(3):166-73.
The polymerase chain reaction (PCR) is a method by which specific sequences of DNA can be copied many times, allowing detailed molecular studies to be performed on as little as a single cell. Numerous and diverse applications of PCR are being developed across all disciplines of diagnostic pathology and research, and no single protocol is appropriate for all situations. Optimising PCR requires a delicate balance between the amplification of specific products and avoiding the production of non-specific products. Each step, from DNA template extraction to cycling times and temperatures, needs to be considered carefully. The aim of this study is to assess which parameters influence DNA amplification efficiency and specificity. The parameters evaluated are the denaturation, annealing and extension temperatures, the number of cycles performed, and the primer, magnesium chloride, dNTP, Taq DNA polymerase and DNA template concentrations. The important parameters for efficient, specific amplification were denaturation time and temperature, stringent annealing temperatures and magnesium chloride concentration. The importance of DNA concentration was found to depend upon the source from which the DNA was extracted.
聚合酶链反应（PCR）是一种能将特定DNA序列多次复制的方法，使得即便仅对单个细胞也能开展详细的分子研究。在诊断病理学和研究的各个学科领域，PCR的应用正在不断拓展且形式多样，不存在适用于所有情况的单一方案。优化PCR需要在特异性产物的扩增与避免非特异性产物的产生之间达成微妙平衡。从DNA模板提取到循环次数和温度，每一个步骤都需要仔细考量。本研究的目的是评估哪些参数会影响DNA扩增效率和特异性。所评估的参数包括变性温度、退火温度和延伸温度、循环次数，以及引物、氯化镁、脱氧核苷三磷酸（dNTP）、Taq DNA聚合酶和DNA模板浓度。实现高效、特异性扩增的重要参数是变性时间和温度、严格的退火温度以及氯化镁浓度。结果发现DNA浓度的重要性取决于所提取DNA的来源。
