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

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.