https://www.nature.com/articles/d41586-024-02280-1 Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Advertisement Nature * View all journals * Search * Log in * Explore content * About the journal * Publish with us * Subscribe * Sign up for alerts * RSS feed 1. nature 2. news 3. article * NEWS * 10 July 2024 Serious errors plague DNA tool that's a workhorse of biology Researchers analysed thousands of laboratory-made plasmids and discovered that nearly half of them had defects, raising questions of experimental reproducibility. By * Katherine Bourzac 1. Katherine Bourzac View author publications You can also search for this author in PubMed Google Scholar * Twitter * Facebook * Email A transmission electron micrograph of genetically engineered DNA plasmids from bacteria that look like closed circles. Plasmids (shown here in a coloured transmission electron micrograph with various genes highlighted) are circular DNA structures used in biology laboratories.Credit: Dr Gopal Murti/SPL Laboratory-made plasmids, a workhorse of modern biology, have problems. Researchers performed a systematic assessment of the circular DNA structures by analysing more than 2,500 plasmids produced in labs and sent to a company that provides services such as packaging the structures inside viruses so they can be used as gene therapies. The team found that nearly half of the plasmids had design flaws, including errors in sequences crucial to expressing a therapeutic gene. The researchers posted their findings to the preprint server bioRxiv last month ahead of peer review^1. The study shines a light on "a lack of knowledge" about how to do proper quality control on plasmids in the lab, says Hiroyuki Nakai, a geneticist at Oregon Health & Science University in Portland who was not involved in the work. He was already aware of problems with lab-made plasmids, but was surprised by the frequency of errors uncovered by the study. There are probably many scientific papers that have been published for which the results are not reproducible owing to errors in plasmid design, he adds. Wasted time Plasmids are popular tools in biology labs because bacteria, including the widely used model organism Escherichia coli, use the structures to store and exchange genes. This means that biologists can make designer plasmids containing various genes of interest, and then coax E. coli to take them up and make lots of copies. [d41586-024] Reproducibility crisis: Blame it on the antibodies Bruce Lahn, chief scientist at VectorBuilder, a company based in Chicago, Illinois, that provides gene-delivery tools, says that he and other biologists have been noticing problems with plasmid quality for years. For example, when Lahn was a professor at the University of Chicago, a graduate student in his lab spent six months trying to reproduce two plasmids that had been reported in the scientific literature. "We didn't think twice about the quality of the plasmids, but then the experiment wouldn't work" because the plasmids contained errors, he says. Now at VectorBuilder, Lahn says he's faced with the issue all the time -- so he decided to evaluate it systematically. When customers submit error-laden plasmids, "it ends up wasting a lot of time", and the extra steps involved in doing quality control add to the cost of producing the plasmids and packaging them into viruses, he says. The VectorBuilder team's analysis found a hodgepodge of errors in the more than 2,500 plasmids it evaluated. Some contained genes that coded for proteins toxic to E. coli, which means that they could slow or stop the growth of the organisms biologists rely on to replicate their plasmids. Others, destined for packaging into viruses, encoded proteins toxic to those viruses. And some contained repetitive DNA sequences that can accumulate mutations inside plasmids. Checking for errors The most rampant errors Lahn and his colleagues found were related to a key gene-therapy tool. Therapies are often packaged into adeno-associated viruses (AAVs), which are mostly harmless and can ferry treatments to cells. When making the plasmids for these AAVs, researchers sandwich a therapeutic gene between sequences called ITRs, which play a crucial part in ensuring that the gene gets packaged into the virus for delivery. In essence, these sequences send a biological signal to cells that says "I belong in this virus". But the team found that about 40% of the AAV plasmids in the study had mutations in the ITR regions that could garble this important message. If researchers were to use these misdesigned plasmids, their gene therapy might not work -- and it could take the scientists a long time to find out why. [d41586-024] Engineered yeast breaks new record: a genome with over 50% synthetic DNA Mark Kay, a paediatrics and genetics specialist at the Stanford School of Medicine in California, has also seen at first hand that plasmid errors can delay lab projects. But he's confident that scientists can spot and fix these errors. He says that gene-therapy researchers are familiar with potential ITR issues, and that errors are unlikely to lead to problems in clinical settings. That's because regulatory agencies such as the US Food and Drug Administration have stringent standards that require researchers to carefully analyse their plasmids before using them in the clinic. Nakai says checking plasmids for errors by sequencing them could alert researchers to the problems highlighted in the study. A few companies, including Plasmidsaurus in Eugene, Oregon, and Elim Biopharmaceuticals in Hayward, California, offer plasmid sequencing for about US$15.00 per sample, says Nakai, who has no financial interest in either company. He also recommends that new lab members spend time learning from experienced plasmid constructors; it's a tedious, artisanal process, he says, but if you get it wrong, it can waste a tremendous amount of time and money. Another way for labs to avert issues is by publicly sharing their plasmid sequences in open-access repositories, says Melina Fan, chief scientific officer of the non-profit organization Addgene in Watertown, Massachusetts. Addgene provides one such repository, Fan says, and it "sequences the deposited plasmids and shares the sequence data via its website for community use". Verification of plasmids is important, she adds. Lahn hopes that his team's analysis will draw researchers' attention to the fact that these workhorse lab tools are often taken for granted. "The health of the tool is not something people question," he says, even though they should. doi: https://doi.org/10.1038/d41586-024-02280-1 References 1. Bai, X. et al. Preprint at bioRxiv https://doi.org/10.1101/ 2024.06.17.596931 (2024). Download references Reprints and permissions Related Articles * [d41586-024] Engineered yeast breaks new record: a genome with over 50% synthetic DNA * [d41586-024] Reproducibility crisis: Blame it on the antibodies * [d41586-024] The immune system can sabotage gene therapies -- can scientists rein it in? * [d41586-024] How gene therapy is emerging from its 'dark age' Subjects * Gene therapy * Scientific community * Synthetic biology * Biological techniques Latest on: Gene therapy Scientists edit the genes of gut bacteria in living mice Scientists edit the genes of gut bacteria in living mice News 10 JUL 24 Streamlined collaboration can boost CRISPR gene therapies for rare diseases Correspondence 02 JUL 24 Hope, despair and CRISPR -- the race to save one woman's life Hope, despair and CRISPR -- the race to save one woman's life News Feature 12 JUN 24 Scientific community The Spinoff Prize 2024 The Spinoff Prize 2024 Outlook 11 JUL 24 Edward C. Stone obituary: physicist who guided Voyager probes to interstellar space Edward C. Stone obituary: physicist who guided Voyager probes to interstellar space Obituary 10 JUL 24 How PhD students and other academics are fighting the mental-health crisis in science How PhD students and other academics are fighting the mental-health crisis in science News Feature 09 JUL 24 Synthetic biology No CRISPR: oddball 'jumping gene' enzyme edits genomes without breaking DNA No CRISPR: oddball 'jumping gene' enzyme edits genomes without breaking DNA Technology Feature 27 JUN 24 Selective haematological cancer eradication with preserved haematopoiesis Selective haematological cancer eradication with preserved haematopoiesis Article 22 MAY 24 Vaccine-enhancing plant extract could be mass produced in yeast Vaccine-enhancing plant extract could be mass produced in yeast News & Views 08 MAY 24 Nature Careers Jobs * Postdoctoral Researcher - Schmidt AI in Science Fellow The University of Toronto now recruiting for the Eric and Wendy Schmidt AI in Science Postdoctoral Fellowship. Valued at $85,000 CDN per year. Toronto (City), Ontario (CA) University of Toronto (U of T) [] * Associate or Senior Editor (Quantum Physics and Quantum Technologies) To help us to build on the success of this journal, we're seeking a researcher with a background in quantum physics. London or Madrid - hybrid working model. Springer Nature Ltd [] * Five industrial PhD students to the Research School in Future Silviculture We are looking for five industrial PhD students to join the Research School in Future Silviculture at the Swedish University of Agricultural Sciences. Umea, Uppsala Swedish University of Agricultural Sciences [] * 'Excellence by Choice' Postdoctoral Programme in Life Science Up to four postdoctoral fellowships within 'Excellence by Choice' Postdoctoral Programme in Life Science at Umea University, Sweden Umea, Sweden Umea University (KBC) [] * Southeast University Future Technology Institute Recruitment Notice Professor openings in mechanical engineering, control science and engineering, and integrating emerging interdisciplinary majors Nanjing, Jiangsu (CN) Southeast University [] You have full access to this article via your institution. Download PDF Related Articles * [d41586-024] Engineered yeast breaks new record: a genome with over 50% synthetic DNA * [d41586-024] Reproducibility crisis: Blame it on the antibodies * [d41586-024] The immune system can sabotage gene therapies -- can scientists rein it in? * [d41586-024] How gene therapy is emerging from its 'dark age' Subjects * Gene therapy * Scientific community * Synthetic biology * Biological techniques Advertisement Sign up to Nature Briefing An essential round-up of science news, opinion and analysis, delivered to your inbox every weekday. Email address [ ] [ ] Yes! Sign me up to receive the daily Nature Briefing email. I agree my information will be processed in accordance with the Nature and Springer Nature Limited Privacy Policy. Sign up * Close Nature Briefing Sign up for the Nature Briefing newsletter -- what matters in science, free to your inbox daily. Email address [ ] Sign up [ ] I agree my information will be processed in accordance with the Nature and Springer Nature Limited Privacy Policy. Close Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing Explore content * Research articles * News * Opinion * Research Analysis * Careers * Books & Culture * Podcasts * Videos * Current issue * Browse issues * Collections * Subjects * Follow us on Facebook * Follow us on Twitter * Subscribe * Sign up for alerts * RSS feed About the journal * Journal Staff * About the Editors * Journal Information * Our publishing models * Editorial Values Statement * Journal Metrics * Awards * Contact * Editorial policies * History of Nature * Send a news tip Publish with us * For Authors * For Referees * Language editing services * Submit manuscript Search Search articles by subject, keyword or author [ ] Show results from [All journals] Search Advanced search Quick links * Explore articles by subject * Find a job * Guide to authors * Editorial policies Nature (Nature) ISSN 1476-4687 (online) ISSN 0028-0836 (print) nature.com sitemap About Nature Portfolio * About us * Press releases * Press office * Contact us Discover content * Journals A-Z * Articles by subject * protocols.io * Nature Index Publishing policies * Nature portfolio policies * Open access Author & Researcher services * Reprints & permissions * Research data * Language editing * Scientific editing * Nature Masterclasses * Research Solutions Libraries & institutions * Librarian service & tools * Librarian portal * Open research * Recommend to library Advertising & partnerships * Advertising * Partnerships & Services * Media kits * Branded content Professional development * Nature Careers * Nature Conferences Regional websites * Nature Africa * Nature China * Nature India * Nature Italy * Nature Japan * Nature Middle East * Privacy Policy * Use of cookies * Your privacy choices/Manage cookies * Legal notice * Accessibility statement * Terms & Conditions * Your US state privacy rights Springer Nature (c) 2024 Springer Nature Limited