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Open Access Highly Accessed Research article

A mechanism for ramified rolling circle amplification

Thomas P Beals*, James H Smith, Raymond M Nietupski and David J Lane

Author Affiliations

Thorne Diagnostics, Beverly, MA, USA

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BMC Molecular Biology 2010, 11:94  doi:10.1186/1471-2199-11-94

Published: 7 December 2010

Abstract

Background

Amplification of single-stranded DNA circles has wide utility for a variety of applications. The two-primer ramified rolling circle amplification (RAM) reaction provides exponential DNA amplification under isothermal conditions, creating a regular laddered series of double-stranded DNA products. However, the molecular mechanism of the RAM reaction remains unexplained.

Results

A RAM reaction model predicts exponential accumulation of a double-stranded DNA product size series, and product-size ratios, that are consistent with observed RAM reaction products. The mechanism involves generation of a series of increasing size intermediate templates; those templates produce RAM products and recursively generate smaller intermediate templates. The model allows prediction of the number of rounds of circular template replication. Real-time RAM reaction data are consistent with the model. Analysis of RAM reaction products shows exponential growth limitation consistent with the model's predictions.

Conclusions

The model provides a rationale for the observed products of the RAM reaction, and the molecular yield among those products. Experimental results are consistent with the model.