Recombinant DNA Technology “Gene Cloning” Gene Cloning
Gene Cloning What is it? • Gene cloning: production of large quantities of a specific, desired gene or section of DNA to produce identical copies • Cloning requires the use of a vector which is a self replicating molecule that can replicate inside the host organism. • The two main kind of vectors are Plasmids and bacteriophages • The Purpose of gene cloning is to yield large quantities of a particular gene or its protein product.
1. Plasmid obtained from bacteria 2.Gene containing foreign DNA is isolated using restriction enzymes 3. Isolated DNA is inserted into plasmid with DNA ligase 4.Plasmids are then inserted back into the bacteria (transformation) 5. Bacteria then multiple, producing identical copies of the “foreign DNA” i.e. Clones Antibiotic resistance gene is also added to plasmid to act as a marker A marker gene is also added to the recombinant DNA Lac-c
Screening Process • Two marker genes an antibiotic resistance gene and a second marker gene Lac-c • Recombinant DNA planted on agar containg x-gal and an antibiotic • Lac-c codes for beta galactosidase enzyme which catalyzes a colourless chemical x-gal to form a blue compound. Thus would appear blue on agar. • If the Lac-c gene is disrupted by the insertion of the isolated gene beta galactosidase enzyme can not be produced. Thus colonies will stay white on agar • bacterial cells on agar containing antibiotic and X-gal any colonies that grow and are white contain our recombinant DNA with our gene of interest.
Preparing the gene of interest • The gene you want to be cloned • Bacteria can’t remove introns so we need to carry out Reverse transcription • mRNA from gene of interest and make DNA using reverse transcriptase, an enzyme which allows a complementary strand to be formed using mRNA as a template.
1. Plasmid obtained from bacteria 3. Isolated DNA is inserted into plasmid with DNA ligase 2.Gene containing foreign DNA is isolated using restriction enzymes 4.Plasmids are then inserted back into the bacteria (transformation) 5. Bacteria then multiple, producing identical copies of the “foreign DNA” i.e. Clones Bacteria then plated onto an agar plate containg X-gal and an antibiotic. Only bacteria that grow are the ones that received wanted DNA
Key points • Prepare gene of interest mRNA to DNA • Restriction enzymes to cut DNA plus plasmid • Marker genes, antibiotic resistance and lac-c are added • DNA ligase to attach isolated DNA with plasmid • Insertion back into host bacterium (transformation) recombinant DNA • Replication • Screening is then carried out to find which cells contain the plasmids with the gene of interest.
Host Bacterium Phage attaches to Bacterium and injects its DNA Bacteriophage Phage uses bacterial metabolism to make more phage particles
Summary • Reproductive cloning for endangered species • Therapeutic cloning technology may some day be used in humans to produce whole organs from single cells or to produce healthy cells to replace degenerative cells • Resistance to disease or improve nutritional quality