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Molecular cloning of African swine fever virus DNA
Abstract:
African swine fever virus DNA (about 170 kbp) was cleaved with the restriction endonuclease EcoRI and most of the resulting 31 fragments were cloned in either the phage vector lambda WES lambda B or the plasmid pBR325. Three fragments were not cloned in those vectors, the largest fragment EcoRI-A (21.2 kbp) and the two crosslinked terminal fragments, EcoRI-K' and D'. Endonuclease SalI cut fragment EcoRI-A into three pieces which were cloned in plasmid pBR322. The two terminal EcoRI fragments were cloned after removal of the crosslinks with nuclease S1 and addition of EcoRI linkers to the fragment ends. The complete library of the cloned fragments accounted for about 98% of ASF virus genome, the missing sequences being those removed by the nuclease S1 in the process of cloning the terminal fragments.
Insights
Researchers created a comprehensive library of African swine fever virus DNA fragments. This library, covering 98% of the genome, aids in understanding the virus and developing control strategies.
Area of Science:
- Virology
- Molecular Biology
- Genomics
Background:
- African swine fever virus (ASFV) poses a significant threat to swine populations globally.
- Understanding the ASFV genome is crucial for developing effective diagnostics and control measures.
Purpose of the Study:
- To construct a complete library of cloned African swine fever virus DNA fragments.
- To facilitate further research into ASFV genetics and molecular mechanisms.
Main Methods:
- ASFV DNA was digested with EcoRI restriction endonuclease.
- Fragments were cloned into lambda WES lambda B phage or pBR325 plasmid vectors.
- Unclonable fragments were further processed and cloned using SalI, S1 nuclease, and EcoRI linkers.
Main Results:
- A library representing approximately 98% of the ASFV genome was successfully generated.
- Most EcoRI fragments were cloned, with special methods used for large and crosslinked terminal fragments.
- The library includes fragments cloned into both phage and plasmid vectors.
Conclusions:
- The generated ASFV DNA library provides a valuable resource for genomic studies.
- This comprehensive library aids in the detailed analysis of the ASFV genome.
- The cloning strategy successfully addressed challenges with large and terminal DNA fragments.