Zazzi M, Romano L, Peruzzi F, Toneatto S, De Milito A, Botta G, Valensin P E
Department of Molecular Biology, University of Siena, Italy.
Mol Cell Probes. 1993 Dec;7(6):431-7. doi: 10.1006/mcpr.1993.1064.
An assessment of optimal conditions for nested primer amplification of low copy number target DNA sequences was made using a human immunodeficiency virus type 1 (HIV-1) model. In this polymerase chain reaction (PCR) strategy, an outer primer pair is first used to amplify the target sequence and a fraction of the amplification product is further amplified with a pair of inner (nested) primers. Several methodological parameters were evaluated, including number of cycles in the first and second step of the reaction, proportion of preamplified material to be used as the template for the second amplification, concentrations of primers, deoxynucleotides, and Taq DNA polymerase in the outer and inner PCR. The two-step PCR required minimal amounts of reaction components and was shown to be highly flexible, resulting in exquisite and specificity over a wide range of technical conditions. Potential drawbacks of this practical and effective amplification procedure are also discussed.
利用1型人类免疫缺陷病毒（HIV-1）模型对低拷贝数靶DNA序列的巢式引物扩增的最佳条件进行了评估。在这种聚合酶链反应（PCR）策略中，首先使用一对外部引物扩增靶序列，然后用一对内部（巢式）引物对部分扩增产物进行进一步扩增。评估了几个方法学参数，包括反应第一步和第二步的循环次数、用作第二次扩增模板的预扩增材料的比例、外部和内部PCR中引物、脱氧核苷酸和Taq DNA聚合酶的浓度。两步PCR所需的反应成分最少，且具有高度灵活性，在广泛的技术条件下都能实现精确性和特异性。本文还讨论了这种实用且有效的扩增方法的潜在缺点。
