ssDNA (Single-Stranded DNA Synthesis)
ssDNA, or single-stranded DNA, is a nucleotide sequence involved in DNA replication and DNA repair. GENEWIZ ssDNA Synthesis service provides up to 10,000 nt of full sequence-verified fragments quickly and affordably. Our ssDNA Synthesis fragments are derived from clonally purified double-stranded DNA (dsDNA), producing the highest quality results possible.
Recent research in CRISPR/Cas9 technology has shown that the use of long ssDNA donor templates greatly enhances the efficiency of homology-directed repair (HDR) enabling researchers to optimize the process of efficiently generating transgenic animal models and cell lines. As the leader in complex gene sequences, you can trust GENEWIZ for sequence-verified ssDNA synthesis for CRISPR gene knock-in, in-vitro transcription, and much more.
What is the difference between dsDNA and ssDNA?
dsDNA stands for double-stranded DNA, which is two complimentary DNA strands forming a helical shape bound by hydrogen bonds. ssDNA stands for single-stranded DNA, and has a lower stability compared to dsDNA. Due to its double-stranded nature and strong hydrogen bond linkage, dsDNA is resistant to formaldehyde (unlike ssDNA).
ssDNA Synthesis Applications
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Antibody Discovery: Engineer customized cell lines or transgenic mouse models to study in vivo immune responses
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Food technology: Modify genomes of agricultural crops to study pathogenic resistance for improved food security
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Cancer Biology: Generate CRISPR gene insertions to study oncogene function for targeted therapeutics
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Biofuels: Precise genome editing to optimize metabolic pathways for biofuel production
CRISPR Gene Knock-In Workflow
Double-stranded breaks are generated through CRISPR/Cas9 editing , then repaired by the endogenous cellular pathways of non-homologous end joining (NHEJ) and HDR. While the HDR pathway has consistently proven successful in copying genetic information via homologous recombination, insertion of exogenous genetic material is a challenge due to the inherent inefficiencies of HDR. Double-stranded DNA has historically been the template of choice for gene insertions, but recent research has shown the superiority of ssODNs as a donor template for HDR. Offering much higher efficiency to insert long sequences with shorter homology arms, ssDNA has become the preferred donor template for this process. GENEWIZ from Azenta now offers the longest (up to 10,000 nt) ssDNA fragments on the market, allowing insertion of long sequences with high efficiency and reduced cellular toxicity or off-target integration compared to dsDNA donors.
ssDNA Advantages for CRISPR Gene Editing
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Lower cellular toxicity compared to dsDNA after cellular delivery
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Low off-target integration for more reliable gene knock-ins
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High specificity knock-in templates for homology directed repair
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High efficiency donors for targeted insertions and gene replacements
Fastest and Longest ssDNA Available
| Length | Available Yield | Turnaround Time |
|---|---|---|
| 151-500 nt | 2, 3, 6, 10, 20, or 40 µg | Starting at 10 Business Days |
| 501 – 2,000 nt | 3, 6, 10, 20, or 40 µg | Starting at 15 Business Days |
| 2,001 – 8,000 nt | 3, 6, 10, 20, or 40 µg | Starting at 20 Business Days |
Please note: 50% off complementary sequence for every order. Custom discount available for customer-supplied dsDNA.
Submit custom inquiries for orders >8,000 nt here.
Single-Stranded DNA Features & Benefits
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Ph.D.-level consultation and support – Our dedicated Project Management team will tailor the order to your exact specifications and support your project from start to finish.
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Advanced capabilities – Standard ssDNA synthesis service ranging from 150 nt to 8000 nt in length, with difficult stretches, like highly repetitive, AT- or GC-rich DNA. For ssDNA over 8000 nt in length, please contact us here.
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AAV control applications – Synthesize ITR at each end of transgene. For the application such as AAV NGS library control, AAV qPCR control or AAV DNA ladder control.
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Quality control – Stringent quality control process with a 100% sequence accuracy guarantee. Residue rate of dsDNA lower than 0.5%.
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Yield flexibility – Choose 2, 3, 6, 10, 20, or 40 µg of lyophilized fragments.
Technical Resources
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Webinar | Understanding Biological Mechanisms to Better Predict the Evolution of Antibiotic Resistance
The rapid and wide-spread evolution of antibiotic resistance is threatening global health. In this webinar, presented by Dr. Mato Lagator, Wellcome Trust, Royal Society Sir Henry Dale Fellow, University of Manchester, you will learn about different approaches to study evolutionary processes that underpin the emergence of resistance. Special focus is given on how these methods can be utilized to improve drug development and longevity.
Deliverables
- Sequence verified
- 2, 3, 6, 10, 20, or 40 µg lyophilized DNA
- Certificate of Analysis (COA) including gel image, sequence trace data with alignment, and sequence files
ssDNA Quality Control
- Sequence confirmation via Sanger sequencing
- Size verification by gel electrophoresis
- S1 nuclease digestion test
Related Publications
- Li H, Beckman KA, Pessino V, et al. Design and specificity of long ssDNA donors for CRISPR-based knock-in. bioRxiv 178905. doi: 10.1101/178905.
- Mason DM, Weber CR, Parola C, et al. High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis. Nucleic Acids Res. (2018);46(14):7436-7449. doi: 10.1093/nar/gky550
- Miura H, Quadros RM, Gurumurthy CB, et al. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc. (2018) 13(1):195-215. doi: 10.1038/nprot.2017.153
- Roth TL, Puig-Saus, C, Yu R, et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559. (2018) 405–409. doi: 10.1038/s41586-018-0326-5.