A Comparison of Methods for the Production of Kilobase-Length Single-Stranded DNA

Chang-Yong Oh,Eric R Henderson

doi:10.3390/dna2010005

Abstract

DNA nanoengineering, in particular, DNA origami has potential applications in a variety of areas including, for example, nanoelectronics, biomedical diagnostics, and therapeutics. To fully realize the potential of DNA self-assembly in these and other areas, methods must be available for economical, scalable, and reliable production of single-stranded DNA (ssDNA) scaffolds from virtually any source. In this review, we will describe the virtues and liabilities of four strategies for generating ssDNA, including Rolling Circle Amplification (RCA), strand-specific exonuclease digestion, chemical denaturation, and asymmetric PCR (aPCR), with suggestions for approaches to optimize the use of each method.

Highlights

Nanoengineering by virtue of DNA-based self-assembly [1–5] is emerging as a platform methodology for addressing a variety of interesting issues ranging from nanoelectronics to biomedical diagnostics and therapeutics [5–10]
Rolling Circle Amplification (RCA) in the presence and absence of single-strand binding protein (SSB) resulted in high molecular weight bands above 10 kb and material retained in the gel wells
In the case of RCA without SSB, the high molecular weight band above 10 kb was fully digested whereas the high molecular weight material in the gel wells remained undigested, suggesting that a significant portion of RCA product in the absence of SSB was double-stranded DNA (dsDNA) with some high molecular weight single-stranded DNA (ssDNA) produced

Summary

Introduction

Nanoengineering by virtue of DNA-based self-assembly [1–5] is emerging as a platform methodology for addressing a variety of interesting issues ranging from nanoelectronics to biomedical diagnostics and therapeutics [5–10]. The use of the M13 scaffold has led to a vast number of advances, it significantly constrains the realization of the full potential of the DNA self-assembly platform. We describe the virtues and liabilities of four relatively economical and simple approaches for producing long ssDNA molecules. These methods are 1, Rolling Circle Amplification (RCA), 2, strand-specific exonuclease digestion, 3, chemical denaturation, and, 4, asymmetric polymerase chain reaction (aPCR)

Methods

Results

Discussion

Conclusion