Improved gap-repair cloning method that uses oligonucleotides to target cognate sequences

Area of Science:

• Molecular Biology
• Yeast Genetics
• Recombinant DNA Technology

Background:

• Yeast gap-repair cloning is a highly efficient method for error-free plasmid construction.
• Existing protocols can be cumbersome and require specific restriction sites for DNA fragment insertion.

Purpose of the Study:

• To describe an optimized protocol for easy and efficient gap-repair cloning in yeast.
• To enhance positive clone identification through a novel screening strategy.

Main Methods:

• Targeting plasmids are constructed using oligonucleotides with upstream/downstream sequences of the target fragment.
• Oligonucleotides generate blunt-end restriction sites for universal DNA fragment compatibility.
• Annealed oligonucleotides are cloned in-frame with a lacZ fragment for blue/white screening in E. coli.

Main Results:

• The modified protocol facilitates efficient cloning of various DNA fragments, including gene fragments (CDC28, CAK1, CIN5, CLB2).
• Approximately 50 bp targeting ends ensure efficient cloning.
• Positive clones are easily identified by blue colony formation on X-Gal media, while successful yeast transformants yield white colonies.

Conclusions:

• This optimized gap-repair cloning method offers a versatile and efficient approach for plasmid construction.
• The integrated lacZ screening simplifies the identification of correctly cloned DNA inserts.
• The protocol's applicability to any DNA fragment, regardless of existing restriction sites, enhances its utility in molecular biology research.