TempliPhi, φ29 DNA polymerase based rolling circle amplification of templates for DNA sequencing

Abstract

We have developed a novel, isothermal DNA amplification strategy that employs φ29 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 φ29 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 φ29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of φ29 DNA polymerase and its use in applications such as amplification of human genomic DNA for genotyping studies is discussed.