Abstract
BackgroundAmplification 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.ResultsA 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.ConclusionsThe 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.
Highlights
Amplification of single-stranded DNA circles has wide utility for a variety of applications
Equal fluorescence with increasing size suggests a decrease in molecule-number that is proportional to the length of the DNA molecule in the band
Because real-time instruments and analytical algorithms were developed for the polymerase chain reaction (PCR), we investigated whether mechanistic differences between PCR and rolling circle amplification (RAM) would affect real-time RAM signal analysis
Summary
Amplification of single-stranded DNA circles has wide utility for a variety of applications. The twoprimer ramified rolling circle amplification (RAM) reaction provides exponential DNA amplification under isothermal conditions, creating a regular laddered series of double-stranded DNA products. Detection of circularized single-stranded DNA (ssDNA) molecules (padlock probes, [2]) has been brought to bear on applications as diverse as SNP detection [3,4,5], miRNA detection [6], and molecular diagnostics [7,8]. In contrast to single primer RCA [12] and the PCR, no detailed and predictive model for the RAM reaction has yet appeared, illustrative diagrams of twoprimer ssDNA circle amplification have been published [11,13]. We present a model of the RAM reaction that accounts for the size and number of doublestranded DNA (dsDNA) product molecules
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