Intra-Molecular Homologous Recombination of Scarless Plasmid
Abstract
:1. Introduction
2. Results
2.1. Intra-HR into Plasmid
2.1.1. Protocol Overview
2.1.2. Primer Design Rule
2.1.3. Plasmid Amplification, Recombination, and Transformation
2.1.4. Transformant Efficiency and Plasmid Accuracy
2.1.5. The Intra-HR Generality
2.2. Comparing with the Two-Step PCR
2.3. Comparing with the ExnaseII Cloning
2.4. Optimal Length of Homology Arm
2.5. Origin of Transformant
3. Discussion
3.1. Advantages
3.2. Limitations
3.3. Scar-Producing Mechanism
4. Materials and Methods
4.1. Materials
4.2. Intra-HR of Plasmid pET20b-AdD
4.2.1. Megaprimer Amplification
4.2.2. Plasmid Amplification
4.2.3. Intra-Molecular HR and Transformation
4.3. Comparing with the Two-Step PCR
4.4. Comparing with the ExnaseII Cloning
4.4.1. Inter-Molecular HR
4.4.2. Intra-Molecular HR
4.5. Optimal Length of Homology Arm
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Method | Enzyme/Reaction | Plasmids | Efficiency (Transformants/ng) | Nicked DNA (ng) | Accuracy (%)/Scarless Plasmids |
---|---|---|---|---|---|
Intra-HR in parallel with the two-step PCR | ExanseII 37°C, 30 min/intra-molecular HR | pET20b-AdD (1′ in Figure 3) | 6 ± 0.5 | 7.2 | 50/1′L1-2,4-5,7 |
pET21a-AdD | 9.8 ± 1.5 | 14.1 | 80/L1-4/7-10 | ||
pET22b-AdD | 37.4 ± 6.8 | 41.3 | 90/L1-4/6-10 | ||
Two-step PCR | T4 DNA ligase 16°C, 16 h/intra-molecular ligation | pET20b-AdD (2′ in Figure 3) | 2.1 ± 0.5 | 16.2 | 0/ |
pET21a-AdD | 25 ± 0.1 | 30.5 | 50/L2,4,6-7,9 | ||
pET22b-AdD | 32.2 ± 5.7 | 27.9 | 70/L1-4,8-10 | ||
ExnaseII cloning | ExanseII 37°C, 30 min/inter-molecular HR | pET20b-AdD with inter-20bp | 5.7 ± 1.1 | nd | 80 |
pET20b-AdD with inter-40bp | 13.5 ± 2.7 | nd | 30 | ||
Intra-HR in parallel with the ExnaseII cloning | ExanseII 37°C, 30 min/intra-molecular HR | pET20b-AdD with intra-20bp | 9.7 ± 1.2 | nd | 80 |
pET20b-AdD with intra-40bp | 13.7 ± 2.3 | nd | 90 | ||
Intra-HR for optimal arm length | ExanseII 37°C, 30 min/intra-molecular HR | pET20b-AdD with intra-10bp | 12.7 ± 0.4 | nd | 90 |
pET20b-AdD with intra-30bp | 14.2 ± 0.2 | nd | 60 | ||
pET20b-AdD with intra-50bp | 11.4 ± 0.9 | nd | 80 |
Primer | Sequence | Tm (°C) |
---|---|---|
AFT | AAGAAGGAGATATACATATG‖ ATTCCTGAACAACCTCAAATGCTCGCAAGT | 72 |
T4R | GTGGTGGTGGTGGTGGTGCTCGAGTAGACCAGTTACCTCATGAAAATC | 61 |
VRPnT | ATTTGAGGTTGTTCAGGAAT‖ CATATGTATATCTCCTTCTTAAAGTTAAACAAAAT | 61 |
AF | p-ATTCCTGAACAACCTCAAATG | 72 |
VRPn | p-CATATGTATATCTCCTTCTTAAAGTTAAACAAAAT | 62 |
AF-L | ATTCCTGAACAACCTCAAATGCTCGCAAGT | 72 |
AR-L | TTCTTTAAATTTATAAAGATTTTTTGAACG | 57 |
ART | TGGTGGTGGTGGTGCTCGAGTTCTTTAAATTTATAAAGATTTTTTGAACG | 57 |
VF | CTCGAGCACCACCACCACC | 65.2 |
VFT | ATCTTTATAAATTTAAAGAACTCGAGCACCACCACCACCA | 73 |
ARP1 | GTTCAGGAATCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGA | 68 |
ARP3 | ACTTGCGAGCATTTGAGGTTGTTCAGGAATCATATGTATATCTCCTTCTTAAAGT | 75 |
ARP5 | ATGCCTTTTTCATCATAAGAACTTGCGAGCATTTGAGGTTGTTCAGGAATCATAT | 76 |
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Liang, Y.; Zhang, Y.; Liu, L. Intra-Molecular Homologous Recombination of Scarless Plasmid. Int. J. Mol. Sci. 2020, 21, 1697. https://doi.org/10.3390/ijms21051697
Liang Y, Zhang Y, Liu L. Intra-Molecular Homologous Recombination of Scarless Plasmid. International Journal of Molecular Sciences. 2020; 21(5):1697. https://doi.org/10.3390/ijms21051697
Chicago/Turabian StyleLiang, Yaping, Yu Zhang, and Liangwei Liu. 2020. "Intra-Molecular Homologous Recombination of Scarless Plasmid" International Journal of Molecular Sciences 21, no. 5: 1697. https://doi.org/10.3390/ijms21051697