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 |
| Published: |
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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| Summary: | 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. |
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| ISSN: | 0736-6205 1940-9818 |