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Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses
Published on: December 29, 2015
Rapid engineering of bacterial artificial chromosomes using oligonucleotides
S Swaminathan1, H M Ellis, L S Waters
1Mouse Cancer Genetics Program, National Cancer Institute-Frederick, Frederick, Maryland 21702-1201, USA.
Genesis (New York, N.Y. : 2000)
|January 3, 2001
Summary
This study introduces a fast, marker-free method for genetically modifying bacterial artificial chromosomes (BACs). It uses synthetic oligonucleotides for precise DNA recombination, enabling efficient BAC engineering for research applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Bacterial artificial chromosomes (BACs) are essential for cloning and manipulating large DNA fragments.
- Traditional BAC modification methods often rely on selectable markers, which can complicate the process.
- A need exists for rapid, marker-free techniques for BAC genetic manipulation.
Purpose of the Study:
- To develop and validate a rapid, marker-free method for genetically manipulating large DNA inserts within BACs.
- To demonstrate the efficiency of using synthetic single-stranded oligonucleotides as targeting vectors for BAC modification.
- To establish a sensitive screening strategy for identifying modified BACs.
Main Methods:
- Utilized synthetic single-stranded oligonucleotides with homology arms for recombining mutations (single-base changes, deletions, insertions) into BACs.
- Eliminated the need for selectable markers during the recombination process.
- Employed pooled bacterial cell cultures and specific PCR amplification for identifying modified BACs without markers.
Main Results:
- Achieved a high frequency of BAC modification, with one recombinant clone per 90-260 electroporated cells.
- Demonstrated the successful introduction of various mutations into BACs using the oligonucleotide recombination method.
- Validated the sensitivity and selectivity of the PCR-based screening approach for identifying desired BAC alterations.
Conclusions:
- The described method offers a rapid and simple approach for BAC manipulation, obviating the need for selectable markers.
- The combination of high targeting efficiency and PCR-based screening significantly streamlines the genetic engineering of BACs.
- This technique provides a valuable tool for researchers working with large DNA constructs in BACs.
