Gene CloningRecombinant DNA Technology“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 enzymes3. 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. ClonesAntibiotic 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

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

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.

Bacteriophage

Host Bacterium

Phage attaches to Bacterium and injects its DNA

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