A mechanism for ramified rolling circle amplification.
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.
Thorne Diagnostics, Beverly, MA, USA. tbeals@verizon.net
Thomas P
TP
Beals
James H
JH
Smith
Raymond M
RM
Nietupski
David J
DJ
Lane
D004247:DNA / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004270:DNA, Circular / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*
D017931:DNA Primers; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004247:DNA