Related Experiment Videos
Use of the lac repressor in constructing sequential deletions and a new sequencing vector
D F Johnson1, D P Nierlich, A J Lusis
1Department of Microbiology, University of California, Los Angeles 90024.
Gene
|September 28, 1990
Summary
This study introduces a method using lac repressor to protect DNA ends, enabling controlled deletions for generating overlapping clones efficiently. This strategy streamlines sequencing of long DNA regions.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Generating overlapping clones is crucial for large-scale DNA sequencing projects.
- Existing methods for creating deletion series can be inefficient and require extensive screening.
Purpose of the Study:
- To develop an efficient strategy for generating a series of overlapping clones for DNA sequencing.
- To utilize the lac repressor-operator system for controlled DNA end protection during exonuclease digestion.
Main Methods:
- Inserting a lac operator sequence into an M13 sequencing vector adjacent to the primer binding site.
- Protecting one end of linearized DNA with lac repressor protein.
- Introducing sequential deletions into the unprotected end using exonuclease III or BAL 31.
- Religating and selecting clones in a timed series to obtain overlapping sequences.
Main Results:
- Demonstrated that lac repressor effectively protects DNA ends containing the lac operator sequence from exonuclease digestion.
- Showcased a method for controlled, sequential introduction of deletions into DNA inserts.
- Facilitated the generation of a timed series of nested deletions, simplifying clone selection.
Conclusions:
- The lac repressor-based DNA end protection strategy significantly enhances the efficiency of generating overlapping clones.
- This method reduces the screening effort required for isolating clones needed for sequencing long DNA regions.
- The technique offers a controlled and streamlined approach for molecular cloning and DNA sequencing applications.