TERMINAL PROTEIN-PRIMED DNA AMPLIFICATION

By using appropriate amounts of four bacteriophage phi 29 DNA replicat ion proteins-terminal protein, DNA polymerase, protein p6 (double-stra nded DNA-binding protein), and protein p5 (single-stranded DNA-binding protein)-it has been possible to amplify limited amounts of the 19,28 5-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 infect ivity 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 esta blish some of the requisites for the development of isothermal DNA amp lification strategies based on the bacteriophage phi 29 DNA replicatio n machinery that are suitable for the amplification of very large (>70 kb) segments of DNA.