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An 'instant gene bank' method for gene cloning by mutant complementation

D Gems¹, A Aleksenko, L Belenky

¹Institute of Genetics, Glasgow University, UK.

Molecular & General Genetics : MGG

|February 1, 1994

Summary

This study introduces an "instant gene bank" method for gene cloning, bypassing traditional library construction. This novel technique efficiently clones genes in vivo using a helper plasmid and fragmented DNA.

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Area of Science:

Molecular Biology
Fungal Genetics
Gene Cloning

Background:

Traditional gene cloning requires constructing and amplifying gene libraries in vitro.
This process is often time-consuming and labor-intensive.

Purpose of the Study:

To develop a novel, efficient in vivo gene cloning method.
To bypass the need for in vitro gene library construction and amplification.

Main Methods:

Simultaneous transformation of mutant Aspergillus nidulans strains with fragmented donor DNA and a helper plasmid.
The helper plasmid contains a fungal origin of DNA replication but lacks a selectable marker.
Gene cloning occurs via ligation or recombination, forming an autonomously replicating plasmid cointegrate in vivo.

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Main Results:

Successfully complemented Aspergillus nidulans niaD, nirA, and argB mutations using Penicillium chrysogenum genomic DNA.
Recovered plasmids from E. coli containing the selected cloned genes.
Demonstrated the cloning of the P. canescens trpC gene using this technique.

Conclusions:

The
instant gene bank
technique offers an efficient alternative for gene cloning.
This method simplifies gene discovery and cloning in fungi.
The technique facilitates the recovery of functional genes from complex genomes.