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A mechanism for ramified rolling circle amplification.


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.

SUBMITTER: Beals TP 

PROVIDER: S-EPMC3017024 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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A mechanism for ramified rolling circle amplification.

Beals Thomas P TP   Smith James H JH   Nietupski Raymond M RM   Lane David J DJ  

BMC molecular biology 20101207


<h4>Background</h4>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.<h4>Results</h4>A RAM reaction model predicts exponential accumulation of a double-stranded DNA product size se  ...[more]

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