Blanco L, Lázaro J M, de Vega M, Bonnin A, Salas M
Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientificas-Universidad Autonoma de Madrid), Universidad Autónoma, Spain.
Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12198-202. doi: 10.1073/pnas.91.25.12198.
By using appropriate amounts of four bacteriophage phi 29 DNA replication proteins--terminal protein, DNA polymerase, protein p6 (double-stranded DNA-binding protein), and protein p5 (single-stranded DNA-binding protein)--it has been possible to amplify limited amounts of the 19,285-bp-long phi 29 DNA molecule by three orders of magnitude after 1 hr of incubation at 30 degrees C. Moreover, the quality of the amplified material was demonstrated by transfection experiments, in which infectivity of the synthetic (amplified) phi 29 DNA, measured as the ability to produce phage particles, was identical to that of the natural phi 29 DNA obtained from virions. The results presented in this paper establish some of the requisites for the development of isothermal DNA amplification strategies based on the bacteriophage phi 29 DNA replication machinery that are suitable for the amplification of very large (> 70 kb) segments of DNA.
通过使用适量的四种噬菌体phi 29 DNA复制蛋白——末端蛋白、DNA聚合酶、p6蛋白（双链DNA结合蛋白）和p5蛋白（单链DNA结合蛋白），在30℃孵育1小时后，已能够将有限量的19285碱基对长的phi 29 DNA分子扩增三个数量级。此外，转染实验证明了扩增产物的质量，在该实验中，合成（扩增）的phi 29 DNA产生噬菌体颗粒的能力所衡量的感染性与从病毒粒子获得的天然phi 29 DNA相同。本文给出的结果确立了基于噬菌体phi 29 DNA复制机制的等温DNA扩增策略发展的一些必要条件，这些策略适用于扩增非常大（>70 kb）的DNA片段。
