Terminal protein-primed DNA amplification.

L Blanco; J M Lázaro; M de Vega; A Bonnin; M Salas

doi:10.1073/pnas.91.25.12198

Home/Archive/PubMed

PubMed · 7991606

Terminal protein-primed DNA amplification.

L Blanco·J M Lázaro·M de Vega·A Bonnin·M Salas

View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

Bacillus Phages DNA Replication DNA, Viral DNA-Binding Proteins DNA-Directed DNA Polymerase Kinetics Models, Structural Nucleic Acid Conformation Transfection Virion Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

L Blanco, J M Lázaro, M de Vega, A Bonnin, M Salas. 1994-12-06. Terminal protein-primed DNA amplification.. https://doi.org/10.1073/pnas.91.25.12198

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Application of Phi29 DNA polymerase in identification and full-length clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf curl Thailand virus.

Tomato plants grown in greenhouses in Thailand developed typical symptoms of a tomato yellow leaf curl Thailand virus (TYLCTHV) infection. After confirmation by ELISA, a Phi29 DNA polymerase approach was chosen for further molecular analysis of TYLCTHV. Total DNA purified from infected tomato leaves was subjected to rolling-circle amplification (RCA) of DNA-A and DNA-B of TYLCVTHV. In addition, a new monopartite geminivirus with a putative recombinant background was identified by RCA and tentatively named tobacco leaf curl Thailand virus (TbLCTHV). To confirm the composition of both geminiviruses, full-length clones were established and used for inoculation of Nicotiana benthamiana by particle bombardment or agroinfection. When TYLCTHV DNA-A and DNA-B were applied together by particle bombardment or agroinfection, severe stunting, yellowing, and leaf curling were observed. Whereas TYLCTHV DNA-A and TbLCTHV revealed no infection after'particle bombardment, similar symptoms in N. benthamiana, like leaf upward curling and yellowing were observed following agroinfection.DNA components of TYLCTHV DNA-A and DNA-B were excised from their respective plasmids, ligated, and amplified by Phi29 DNA polymerase. The ability of viral concatamere inoculation was evaluated in particle co-bombardment experiments on N. benthamiana. Thus, particle bombardment of RCA-derived multimeric products proved to be at least as effective as inoculation with a partial repeat construct and tenfold as effective as inoculation with excised unit-lengths of DNA-A and DNA-B of TYLCVTHV when using each DNA component in an amount of 5 ng.

Bacillus Phages↗

PubMed2007

Indirect detection of Bacillus anthracis using real-time PCR to detect amplified gamma phage DNA.

Typical real-time PCR methods used to identify Bacillus anthracis do not distinguish between viable and non-viable spores, which would be critical in any first response and remediation scenarios. This study combined both real-time PCR, using primers specifically designed for gamma phage, with the highly specific gamma phage amplification into one simple assay to indirectly detect Bacillus anthracis. Since the amplification of gamma phage only occurs in the presence of a suitable host, the detection of increasing concentrations of progeny gamma phage DNA using real-time PCR implies the presence of viable Bacillus anthracis cells. This method detected a starting Bacillus anthracis concentration of 207 cfu/mL, equivalent to less than one cell in 20 microL, in less than 5 h.

Bacillus Phages↗

PubMed2007

Structural and thermodynamic principles of viral packaging.

Packaging of genetic material inside a capsid is one of the major processes in the lifecycle of bacteriophages. To establish the basic principles of packing double-stranded DNA into a phage, we present a low-resolution model of bacteriophage varphi29 and report simulations of DNA packaging. The simulations show excellent agreement with available experimental data, including the forces of packaging and the average structures seen in cryo-electron microscopy. The conformation of DNA inside the bacteriophage is primarily determined by the shape of the capsid and the elastic properties of DNA, but the energetics of packaging are dominated by electrostatic repulsions and the large entropic penalty associated with DNA confinement. In this slightly elongated capsid, the DNA assumes a folded toroidal conformation, rather than a coaxial spool. The model can be used to study packaging of other bacteriophages with different shapes under a range of environmental conditions.

Bacillus Phages↗