Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation

doi: 10.1073/pnas.012372799

Figure Lengend Snippet: Pfu P45 enhances PCRs using archaeal DNA polymerases. Native P45 (0, 1, 5, or 10 ng) was added to amplifications of 0.9-kb and 1.9-kb genomic targets. PCRs were carried out with 2.5 units of Pfu DNA polymerase or 1 unit of Vent R , Deep Vent R , or Taq DNA polymerase in each enzyme's optimal PCR buffer.

Article Snippet: Improvements in the performance of Pfu DNA polymerase have been achieved by increasing PCR extension times (2 min/kb of target; Stratagene Pfu DNA polymerase manual), incorporating additives such as DMSO ( , ), and mixing Pfu with Taq DNA polymerase ( ).

Techniques: Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation

doi: 10.1073/pnas.012372799

Figure Lengend Snippet: Human dUTPase exhibits PCR-enhancing activity. A 5.2-kb target was amplified with 5 units of Pfu DNA polymerase. At each annealing step (58°C), 0.5 μl of the following was added: buffer (lane 1) or human dUTPase preparation, undiluted (lanes 2 and 3), or diluted 10 −1 (lanes 4 and 5).

Article Snippet: Improvements in the performance of Pfu DNA polymerase have been achieved by increasing PCR extension times (2 min/kb of target; Stratagene Pfu DNA polymerase manual), incorporating additives such as DMSO ( , ), and mixing Pfu with Taq DNA polymerase ( ).

Techniques: Polymerase Chain Reaction, Activity Assay, Amplification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation

doi: 10.1073/pnas.012372799

Figure Lengend Snippet: Pfu P45 improves amplification of long targets by Pfu DNA polymerase. PCRs were performed with Taq or Pfu DNA polymerase, in the absence or presence of P45, in each enzyme's recommended buffer. The following PCR products were amplified: lanes 1–4, 7–10, and 13–16, 0.9- to 5.2-kb fragments of the human α-1-antitrypsin gene; lanes 5, 11, and 17, 8-kb fragment from mouse genomic DNA; and lanes 6, 12, and 18, 14-kb fragment from λ phage DNA. PCR amplifications were conducted by using 2.5 units (lanes 4, 10, and 16) or 5 units of each enzyme and an extension time of 1 min/kb of target.

Article Snippet: Improvements in the performance of Pfu DNA polymerase have been achieved by increasing PCR extension times (2 min/kb of target; Stratagene Pfu DNA polymerase manual), incorporating additives such as DMSO ( , ), and mixing Pfu with Taq DNA polymerase ( ).

Techniques: Amplification, Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation

doi: 10.1073/pnas.012372799

Figure Lengend Snippet: PCR-enhancing activity from Pfu . Fractions eluted from heparin-Sepharose ( A ) or Sephacryl S-200 ( B ) columns were added to PCRs using Pfu DNA polymerase. ( A ) A 6.6-kb (lanes 1–8) or 5.2-kb (lanes 9–10) genomic target was amplified in the presence (+) of the following amount of eluate: lanes 1, 3, and 9, none (−); lanes 2, 4, 5, and 10, 1 μl of undiluted; lane 6, 1 μl of diluted 10 −1 ; lane 7, 1 μl of diluted 10 −3 ; and lane 8, 1 μl of diluted 10 −5 . As negative controls, either Pfu DNA polymerase ( A , lanes 1 and 2) or genomic DNA ( A , lanes 3 and 4) was omitted. The 6.6-kb PCR system produces a second nonspecific band (lower band in lanes 5 and 6) irrespective of Pfu P45 addition. ( B ) The following amount (ng) of S-200-purified protein was added (+) to amplifications of an 8-kb genomic target: lane 1, none (−); lane 2, 0.1; lane 3, 0.2; lane 4, 0.5; lane 5, 1; lane 6, 5; lane 7, 25; lane 8, 100; or lane 9, 200.

Article Snippet: Improvements in the performance of Pfu DNA polymerase have been achieved by increasing PCR extension times (2 min/kb of target; Stratagene Pfu DNA polymerase manual), incorporating additives such as DMSO ( , ), and mixing Pfu with Taq DNA polymerase ( ).

Techniques: Polymerase Chain Reaction, Activity Assay, Amplification, Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Comparative assessment of virulence of recombinant vaccinia viruses expressing IL-2 and IL-15 in immunodeficient mice

doi: 10.1073/pnas.081080298

Figure Lengend Snippet: Quantitative assessment of viral DNA in tissues of infected mice. Nude mice were inoculated i.v. with 10 6 pfu of the respective recombinant vaccinia viruses, and tissues were harvested 72 h after infection. Genomic DNA was then isolated from lung (lanes 1), spleen (lanes 2), brain (lanes 3), and liver (lanes 4). Each sample represents tissues pooled from three animals, and the recovery of DNA was first standardized to the cytochrome- c oxidase II gene content in each sample before PCR amplification of the β-galactosidase gene.

Article Snippet: Five micrograms of cellular DNA was used in PCR amplification of the Escherichia coli β-galactosidase DNA sequences with Pfu Turbo DNA polymerase (Stratagene).

Techniques: Infection, Mouse Assay, Recombinant, Isolation, Polymerase Chain Reaction, Amplification