Detailed Studies of Activity of Gene of protein coded by unknown gene

Basically there is an unknown Gene that makes up a Protein with specific function and by deleting that part of the gene and leaving the rest part of the gene which is still performing that function, so to check whether that gene is still performing all the functions right or not....

This is done through the process call Site Directed Mutagenesis or Invitro Mutagenesis.

• So After Mutagenesis, the gene is inserted in the host 
• Afterwards via Homologous recombination Gene Replaces the existing copy of gene
• Still we have no idea what cells have undergone Homologous Recombination.
• So we use a Marker's Gene [ Antibiotic Resistant ]
• If marker gene is present, so will be the mutated gene
• But now the problem is if the That Marker gene is incorporated in genome instead of mutated

so we use a system called a Two step Gene Replacement

• First two Target gene is replaced by Marker Gene on its own.
• The Cells which undergo Recombination are identified by selecting Marker Gene phenotype.
• There cell's are then used in 2nd step where Marker gene is replaced by Mutated Gene.

Afterwards the success is monitored by the looking for the cells that have lost the Marker gene phenotype.

Now the cells contain the Mutated gene and their phenotype can be examined to verify the effect of direct mutated Gene.