TempliPhi, ⌽29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing

Abstract

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.