Multi-enzymatic systems synergize new RCA technique amplified super-long dsDNA from DNA circle

Abstract

Background: In the field of DNA amplification, there are great challenges in the effectively amplify of long-chain amplification, especially amplification up to several hundred kb level.

Results: A novel technique for the unbiased whole genome amplification from a thimbleful of DNA circles, such as low as 10 ng/ 10 μL of the circular cpDNA or low as 5 ng/ 10 μL of the plasmid, is developed, which can amplify an abundance of the whole genome sequences. Specifically, the new technique that combines rolling-amplification and triple-enzyme system presents a tightly controlled process of a series of buffers/reactions and optimized procedures, that applies from the primer-template duplexes to the Elution step. The result of this technique provides a new approach for extending RCA capacity, where it can reach 200 kb from the circular cpDNA amplification and 150 kb from the plasmid DNA amplification, that demonstrates superior breadth and evenness of genome coverage, high reproducibility, small amplification bias with the amplification efficiency.

Significance and novelty: This new technique will develop into one of the powerful tools for isothermal DNA amplification in vitro, genome sequencing/analysis, phylogenetic analysis, physical mapping, and other molecular biology applications.

Keywords: DNA circles; Rolling-amplification; Triple-enzyme system; Whole genome sequences.