Molecular Genetics 

• Complementation of a mutant phenotype in an easily transformable organism can be used to obtain, from a different organism, a gene equivalent to the mutant gene. The mutant is transformed with a library of the target organism. Transformants whose mutant phenotype is corrected contain the target gene.
• When complementation is not feasible, tagging mutagenesis can be used.
• Seeds of Arabidopsis are transformed at high efficiency with T-DNA using Agrobacteria. Resulting transformants are germinated and plantlets screened for mutant phenotypes. Top
• DNAs such as transposable elements and T-DNA that integrate into the chromosomal DNA can create mutations by interruption of the gene in which they insert. Top
• Further genetic manipulation may be needed after the initial tagging event to assure that the tag is indeed associated with the mutant phenotype. Top
• When a transposable element transposes, it often integrates itself in genes, causing mutations that can be recognized by phenotype. Top
• A genomic library is made from DNA of the tagged mutant organism. Top
• The genomic library of the tagged mutant organism is screened by hybridization with the tagging agent as probe. Top
• DNA of the tagged mutant organisms is digested with a restriction enzyme that does not cleave within the tagging agent. The fragments are circularized by ligation at dilute concentrations and used to transform E. coli. Top
• Oligonucleotides complementary to the ends of the tagging agent and with their 3'-ends pointing away from the agent are used to amplify the sequences flanking the tagging agent.
• Positive clones obtained from a rescued plasmid, inverse PCR or a tagged genomic library can be used as nucleic acid hybridization probes to screen a library of genomic DNA to identify clones containing the wild type gene. Top
• Positively reacting genomic library clones need to be further examined to :  
• Identify where on the clone the gene is located.
• Prove that the clone actually contains the gene.

Further Information

• In a variant of tagging mutagenesis, a population of tags is used to generate the mutants.  Each tag differs from the others in a pair of 20mer oligonucleotides that serve as bar codes.  The population of mutants is subjected to a selective pressure.  The bar codes in the survivors are compared with those in the initial population.  Any missing bar codes must derive from a mutation in a gene required to deal with the selective pressure.  The presence of the bar code allows its isolation from the initial population (ref).