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 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 (13 g) 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 L 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.

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

“…To determine the accuracy of DNA amplification by B35DNAP, we circularized digested samples from MDA experiments and subjected them to white/blue colony screening (Materials and Methods). As shown in Table 1, the average error rate was 5.1 ± 1.7 × 10 −6 , similar to that of Φ29DNAP and commercially available PCR polymerases (26,33,34). We also sequenced several mutant (white) clones to determine which type of mistake was the most frequent.…”

“…This result indicates that most of the amplification products were dsDNA tandem repeats of the original plasmid. Importantly, B35DNAP was able to amplify a minimal DNA input of 1-2.5 ng (lanes 6 and 7), similar to that routinely used for Φ29DNAP-mediated MDA of plasmid DNA (26,32). To determine the accuracy of DNA amplification by B35DNAP, we circularized digested samples from MDA experiments and subjected them to white/blue colony screening (Materials and Methods).…”

“…18,20,24). Φ29DNAP is a highly faithful enzyme (25,26) able to generate very long DNA molecules (27), coupling DNA synthesis and strand displacement. Φ29, along with other protein-primed genome replication phages, such as PRD1 or Cp-1 (28,29), can undergo a lytic cycle only after infection of the host cell occurs.…”

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

“…Use of single-stranded M13 DNA as template gave a rolling-circle mode of replication, in which the polymerase repeatedly copied around the circular template via its strand displacement activity, yielding a product of concatenated M13 repeats. The DNA polymerase's associated 3 0 -5 0 exonuclease proofreading activity results in a low intrinsic error rate of 10 À6 -10 À7 (Watabe et al, 1984;Blanco and Salas, 1985) and the accumulation of mutations in MDA products at a Multiple displacement amplification and microbial ecology EK Binga et al rate of only 10 À5 -10 À6 (Esteban et al, 1993;Nelson et al, 2002), nearly 1000-fold less than for PCR using the Taq DNA polymerase (Dunning et al, 1988;Saiki et al, 1988). The first use of MDA for amplifying whole genomes targeted human DNA (Dean et al, 2002) and as few as 90 copies of the genome (0.3 ng DNA) yielded more than 30 mg of product.…”

Rolling circle amplification of metazoan mitochondrial genomes

“…RCA has been used in the ampliWcation of microbial genomes, for the ampliWcation of signal from probes, and in plasmid ampliWcation for sequencing (Dean et al, 2001;Hawkins et al, 2002;Lizardi et al, 1998;Nelson et al, 2002). The beneWts of RCA lie in its universal priming conditions; RCA employs numerous random short primers, which anneal at many sites to a DNA template.…”