topic 8 Gene Technology

making recombinant DNA

1. gene is identified & isolated by restriction enzyme

2. restriction endonuclease cuts DNA strand at specific sites to leave sticky ends

3. a plasmid is cut with the same restriction endonuclease to leave complementary sticky ends

4. gene is inserted into the plasmid and DNA ligase joins the two together

5. forming a recombinant plasmid

Inserting Recombinant DNA into cells

1. Plasmid introduced to host bacterial cell forming a (GM) transformed cell

2. Transformed Cell Production: host cell takes up recombinant plasmid, and the produces the protein coded by inserted gene

3. Marker gene inserted into plasmid to distinguish transformed cells

4. Replica plating

   • A filter paper is pressed onto the original (master) plate to pick up colonies, then pressed onto a second agar plate

Microarray Analysis

1. mRNA is extracted and converted into cDNA using reverse transcriptase

2. The cDNA is labeled with fluorescent markers and applied to a microarray with gene probes

3. It binds to matching probes (hybridization), unbound cDNA is washed off, and fluorescence shows which genes are active in the sample

GM Microorganisms

1. gene is isolates and cut out with restriction endonuclease enzyme

2. plasmid is cut with the same restriction endonuclease to leave complementary sticky ends

3. gene is inserted into the plasmid and DNA ligase joins the two together

4. the vector (plasmid) is used to introduce the gene into the host cell

5. the modified host cell (microorganism) is grown in large fermenters containing nutrients enabling them to multiply and produce large quantities of the new protein

GM Plants

1. ti-plasmid is extracted from A. tumefaciens

2. gene to be carried to the plant is inserted into plasmid that’s returned to bacteria

3. plant is infected with the modified bacteria and the GM part becomes part of the plant chromosome

4. a new plant can be grown from the tumor cell released by bacteria