TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing
We have developed a novel, isothermal DNA amplification strategy that employs ⌽29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi™ DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in...
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| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2002-06-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/jun0208 |
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| author | John R. Nelson Yuyang Christine Cai Theresa L. Giesler Joseph W. Farchaus Shanmuuga T. Sundaram Maria Ortiz-Rivera Lou P. Hosta Peter L. Hewitt J. Anthony Mamone C. Palaniappan Carl W. Fuller |
| author_facet | John R. Nelson Yuyang Christine Cai Theresa L. Giesler Joseph W. Farchaus Shanmuuga T. Sundaram Maria Ortiz-Rivera Lou P. Hosta Peter L. Hewitt J. Anthony Mamone C. Palaniappan Carl W. Fuller |
| author_sort | John R. Nelson |
| collection | DOAJ |
| description | We have developed a novel, isothermal DNA amplification strategy that employs ⌽29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi™ DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using ⌽29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 107-fold amplification. Large amounts of product (1−3 μg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 μL of a saturated overnight culture. Additionally, the presence of an associated proofreading function within the ⌽29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of ⌽29 DNA polymerase and its use in applications such as amplification of human genomic DNA for genotyping studies is discussed. |
| format | Article |
| id | doaj-art-5ef2bb8840cb47b39c5df2a3ae002e1b |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2002-06-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-5ef2bb8840cb47b39c5df2a3ae002e1b2025-08-20T02:25:55ZengTaylor & Francis GroupBioTechniques0736-62051940-98182002-06-01326SS44S4710.2144/jun0208TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA SequencingJohn R. Nelson0Yuyang Christine Cai1Theresa L. Giesler2Joseph W. Farchaus3Shanmuuga T. Sundaram4Maria Ortiz-Rivera5Lou P. Hosta6Peter L. Hewitt7J. Anthony Mamone8C. Palaniappan9Carl W. Fuller101Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USA1Genomics Reagents Department, Amersham Biosciences, Piscataway, NJ, USAWe have developed a novel, isothermal DNA amplification strategy that employs ⌽29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi™ DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using ⌽29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 107-fold amplification. Large amounts of product (1−3 μg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 μL of a saturated overnight culture. Additionally, the presence of an associated proofreading function within the ⌽29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of ⌽29 DNA polymerase and its use in applications such as amplification of human genomic DNA for genotyping studies is discussed.https://www.future-science.com/doi/10.2144/jun0208 |
| spellingShingle | John R. Nelson Yuyang Christine Cai Theresa L. Giesler Joseph W. Farchaus Shanmuuga T. Sundaram Maria Ortiz-Rivera Lou P. Hosta Peter L. Hewitt J. Anthony Mamone C. Palaniappan Carl W. Fuller TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing BioTechniques |
| title | TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing |
| title_full | TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing |
| title_fullStr | TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing |
| title_full_unstemmed | TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing |
| title_short | TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing |
| title_sort | templiphi ⌽29 dna polymerase based rolling circle amplification of templates for dna sequencing |
| url | https://www.future-science.com/doi/10.2144/jun0208 |
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