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Single-Stranded DNA (ssDNA) Synthesis

Single-stranded DNA (ssDNA) fragments are important research tools in the molecular biology field. ssDNA can be used in CRISPR gene therapy, diagnostic assays, DNA nanotechnology and many other applications. ssDNA oligonucleotides are usually produced via well-established chemical solid-phase DNA synthesis on polymeric supports. There is one advantage that almost all type of nucleotide analog can be introduced at well-defined positions. Moreover, ssDNA can increase the likelihood of homology-directed repair with reduced cellular toxicity.

Efficient ssDNA synthesis on the 10+ kb-scale is a major requirement for numerous biotechnology applications including templated homology-directed repair for genome editing, systems-scale gene synthesis and cloning, and scaffolded DNA origami. Nevertheless, as the length is extended, the expected yields will diminish to some degree. Moreover, chemical synthesis is currently limited to approximately 98% incorporation efficiency for each base addition, and to the production of ssDNA oligonucleotides which is only up to only 200 bases. To counter the diminished yield, Creative Biogene has developed enzymatic synthesis methods, allowing us to produce single-strand DNA more than 10,000 nt in some cases, while 3000-5000 nt is common.

ssDNA Synthesis Services at Creative Biogene

Creative Biogene applies purely enzymatic synthesis to generate ssDNA greater than 10 kilobases (kb) using asymmetric polymerase chain reaction (aPCR), and illustrate the incorporation of diverse modified nucleotides for therapeutic and theranostic applications.

aPCR offers the direct synthesis of ssDNA from an underlying dsDNA template, independent of biological replication sequences, and has been applied to generate ssDNA ranging from several hundred to several thousand nucleotides in length. aPCR is different from traditional PCR by having one primer (the forward primer) in molar excess over the second primer (the reverse primer). This method has previously been applied to short ssDNA synthesis for aptamers and gene detection, and more recently to kb-scale ssDNA for scaffolded DNA origami. Nevertheless, previous work was limited to 3.3 kb owing to low enzyme processivity. Here, we overcome this limitation to achieve 10+ kb length ssDNA.

ssDNA production by aPCR. Figure 1. ssDNA production by aPCR.


  • CRISPR-mediated gene knock-in
  • In vitro transcription
  • Systems-scale gene synthesis and cloning
  • DNA aptamer production
  • SNP detection
  • Subtractive hybridization


  • Ready-to-use long ssDNA donor templates up to 10 kb in length
  • Verification by sequencing
  • Increasing the likelihood of homology-directed repair with reduced cellular toxicity
  • Modifications available

Creative Biogene’s ssDNA synthesis services provide fast and affordable, sequence-verified long ssDNA fragments. As the leader in working with complex gene sequences, we can deliver sequence-verified fragments for CRISPR-mediated gene knock-in, in vitro transcription and many other applications. If you have specific requirements, please feel free to contact us.


  1. Veneziano R, et al. In vitro synthesis of gene-length single-stranded DNA. Scientific Reports, 2018, 8.

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