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Rolling circle amplification shows a sinusoidal template length-dependent amplification bias.

Bastian Joffroy¹, Yavuz O Uca¹, Domen Prešern²

¹Center for Advancing Electronics Dresden (cfaed), Technische Universität Dresden, 01062 Dresden, Germany.

Nucleic Acids Research

|December 14, 2017

Summary

DNA template length impacts rolling circle amplification (RCA) efficiency, a nucleic acid amplification method. This bias, linked to B-DNA periodicity, arises from DNA fraying, not end stacking, influencing polymerization rates.

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Area of Science:

Molecular Biology
Biophysics
Biotechnology

Background:

DNA's biophysical properties, including persistence length, are crucial for biological processes.
DNA circularization efficiency exhibits periodicity related to helical turns, attributed to end stacking.
Small DNA circles serve as templates for rolling circle amplification (RCA), a robust nucleic acid amplification technique.

Purpose of the Study:

To investigate the template length-dependent amplification efficiency bias in RCA.
To elucidate the underlying mechanism of this bias, particularly in the context of DNA structure and polymerase activity.

Main Methods:

Analysis of rolling circle amplification (RCA) efficiency across various DNA template lengths.
Coarse-grained molecular dynamics simulations to model DNA behavior at the polymerase fork.
Investigation of DNA end-fraying probabilities and their correlation with amplification efficiency.

Main Results:

A significant template length-dependent bias in RCA amplification efficiency was discovered, mirroring B-DNA periodicity.
This bias could not be explained by DNA end stacking, as polymerase activity inhibits it.
Molecular dynamics simulations indicated that varying fraying probabilities of downstream base pairs correlate with amplification efficiency.

Conclusions:

The observed RCA bias is primarily driven by template length-induced variations in DNA fraying probability.
Increased strain-promoted fraying of DNA ends can enhance the polymerization rate during RCA.
This finding offers new insights into DNA-polymerase interactions and amplification mechanisms.