Principles of genetic technology

Need to know

What you need to know

• **1. Isolation of the Gene of Interest**: The desired gene is identified and isolated from the source organism's DNA. This can be done using restriction enzymes to cut it out from the genome, or by using reverse transcriptase to create a complementary DNA (cDNA) copy from an mRNA template. Using mRNA is advantageous as it has already had the non-coding introns removed.
• **2. Insertion into a Vector**: The isolated gene is inserted into a vector (e.g., a plasmid). The same restriction enzyme is used to cut both the gene and the plasmid, creating complementary 'sticky ends'. The gene anneals to the plasmid, and DNA ligase is used to form permanent phosphodiester bonds, creating a recombinant plasmid.
• **3. Transformation**: The recombinant vector is introduced into a host organism (usually bacteria like *E. coli* or yeast). This process is called transformation. Methods to increase the permeability of the host cell membrane, such as heat shock or electroporation, are often used.
• **4. Identification and Selection**: Not all host cells will successfully take up the vector. Marker genes (e.g., for antibiotic resistance or fluorescence) included in the vector are used to identify and select the transformed cells. For example, if the plasmid carries a gene for ampicillin resistance, only transformed bacteria will grow on a medium containing ampicillin.
• **5. Expression and Harvesting**: The transformed host cells are cultured in large quantities (e.g., in a fermenter). Under the right conditions, they will express the inserted gene, producing the desired protein (e.g., human insulin). The protein is then extracted, purified, and harvested.

Explanation

Principles of genetic technology

1. **1. Isolation of the Gene of Interest**: The desired gene is identified and isolated from the source organism's DNA. This can be done using restriction enzymes to cut it out from the genome, or by using reverse transcriptase to create a complementary DNA (cDNA) copy from an mRNA template. Using mRNA is advantageous as it has already had the non-coding introns removed.
2. **2. Insertion into a Vector**: The isolated gene is inserted into a vector (e.g., a plasmid). The same restriction enzyme is used to cut both the gene and the plasmid, creating complementary 'sticky ends'. The gene anneals to the plasmid, and DNA ligase is used to form permanent phosphodiester bonds, creating a recombinant plasmid.
3. **3. Transformation**: The recombinant vector is introduced into a host organism (usually bacteria like *E. coli* or yeast). This process is called transformation. Methods to increase the permeability of the host cell membrane, such as heat shock or electroporation, are often used.
4. **4. Identification and Selection**: Not all host cells will successfully take up the vector. Marker genes (e.g., for antibiotic resistance or fluorescence) included in the vector are used to identify and select the transformed cells. For example, if the plasmid carries a gene for ampicillin resistance, only transformed bacteria will grow on a medium containing ampicillin.