IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction
Abstract Background Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either i...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-12-01
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| Series: | Plant Methods |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13007-022-00965-0 |
| _version_ | 1811302368422133760 |
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| author | Jonas De Saeger Jihae Park Kai Thoris Charlotte De Bruyn Hoo Sun Chung Dirk Inzé Stephen Depuydt |
| author_facet | Jonas De Saeger Jihae Park Kai Thoris Charlotte De Bruyn Hoo Sun Chung Dirk Inzé Stephen Depuydt |
| author_sort | Jonas De Saeger |
| collection | DOAJ |
| description | Abstract Background Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. Results Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. Conclusions We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology. |
| first_indexed | 2024-04-13T07:26:51Z |
| format | Article |
| id | doaj.art-51cb82e7afc0417d8d173f698735733d |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-04-13T07:26:51Z |
| publishDate | 2022-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-51cb82e7afc0417d8d173f698735733d2022-12-22T02:56:27ZengBMCPlant Methods1746-48112022-12-0118111310.1186/s13007-022-00965-0IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reactionJonas De Saeger0Jihae Park1Kai Thoris2Charlotte De Bruyn3Hoo Sun Chung4Dirk Inzé5Stephen Depuydt6Laboratory of Plant Growth Analysis, Ghent University Global CampusLaboratory of Plant Growth Analysis, Ghent University Global CampusLaboratory of Plant Growth Analysis, Ghent University Global CampusLaboratory of Plant Growth Analysis, Ghent University Global CampusDepartment of Plant Biotechnology and Bioinformatics, Ghent UniversityDepartment of Plant Biotechnology and Bioinformatics, Ghent UniversityLaboratory of Plant Growth Analysis, Ghent University Global CampusAbstract Background Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. Results Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. Conclusions We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.https://doi.org/10.1186/s13007-022-00965-0Copy number determinationCompetitive PCRT-DNA insertionsIMPLANTGenetically modified plants |
| spellingShingle | Jonas De Saeger Jihae Park Kai Thoris Charlotte De Bruyn Hoo Sun Chung Dirk Inzé Stephen Depuydt IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction Plant Methods Copy number determination Competitive PCR T-DNA insertions IMPLANT Genetically modified plants |
| title | IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction |
| title_full | IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction |
| title_fullStr | IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction |
| title_full_unstemmed | IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction |
| title_short | IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction |
| title_sort | implant a new technique for transgene copy number estimation in plants using a single end point pcr reaction |
| topic | Copy number determination Competitive PCR T-DNA insertions IMPLANT Genetically modified plants |
| url | https://doi.org/10.1186/s13007-022-00965-0 |
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