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Isolating Interaction-Null/Impaired Mutants Using the Yeast Two-Hybrid Assay

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Published on: December 29, 2023

Analysis of isolated YAC clones.

D D Chaplin¹, B H Brownstein

¹Howard Hughes Medical Institute and Washington, University School of Medicine, St. Louis, Missouri, USA.

Current Protocols in Molecular Biology

|February 12, 2008

Summary

This unit details protocols for analyzing and manipulating yeast artificial chromosome (YAC) clones using the pYAC4 vector. It covers DNA purification, size assessment, Southern blotting, and end-fragment recovery for genomic analysis.

Area of Science:

Molecular Biology
Yeast Genetics
Genomics

Background:

Yeast artificial chromosomes (YACs) are crucial for cloning large DNA fragments.
Standardized protocols are needed for the analysis and manipulation of isolated YAC clones.

Purpose of the Study:

To provide a comprehensive set of protocols for the analysis and manipulation of isolated YAC clones.
To detail methods for YAC DNA purification, characterization, and end-rescue.

Main Methods:

Culturing and storage of YAC-containing yeast strains.
Purification of YAC DNA for size assessment and Southern blotting.
Preparation of yeast chromosomes in agarose plugs for pulsed-field gel electrophoresis (PFGE).
Recovery of YAC insert end-fragments for probe generation.

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Subcloning of YAC inserts into cosmid or lambda vectors.

Main Results:

Established protocols for handling YAC clones.
Demonstrated methods for assessing YAC integrity and size.
Provided techniques for end-fragment isolation and subcloning for further analysis.

Conclusions:

The described protocols enable robust analysis and manipulation of YAC clones.
These methods facilitate genomic research, including chimerism detection and chromosome walking.
The protocols support higher-resolution analysis through subcloning.