- Procedure that allows DNA fragments or genes to be copied
- Use a restriction enzyme to cut up the foreign DNA that contains a gene to be copied. The restriction enzyme produces multiple fragments of foreign DNA with sticky ends
- Use the same restriction enzyme to cut up the DNA of a cloning vector. This produces the same strictly ends in both foreign DNA and cloning vector: DNA molecule that can carry foreign DNA into a host cell and be replicated there
- Plasmid is a common cloning vector bcuz can be introduced into bacteria for transformation
- Using a plasmid that has one restriction site for restriction enzyme can help with identification of the copied gene
- ampR gene: gives bacterial resistance against antibiotic ampicillin
- GFP gene: makes bacteria fluorescence green
- lacZ gene: codes for enzyme that breaks down lactose
- Also breaks down artificially made X-gal
- The one restriction site for the restriction enzyme occurs in within the lacZ gene
- Mix the cut foreign DNA with cut plasmids. This allows base-pairing at the sticky end
- Apply DNA ligase to stabilize attachments and close up the backbone. Forms recombinant plasmids (some plasmids will not pair)
- Mix plasmids with bacteria to allow transformation. Some of the bacteria will absorb the plasmids (transformation)
- Not all bacteria will be competent enough to take up trait and express traits associated with gene
- Competent: change in structure and permeability of cell membrane
- Grow the transformed bacteria in the presence of ampicillin and X- gal.
- Only bacteria that have absorbed a plasmid (transformed bacteria) will grow in presence of ampicillin bcuz contain the resistant genel; they will also be white bcuz lack functioning lacZ gene (foreign DNA was inserted within lacZ gene of the plasmid, making gene dysfunctional )
- Purposes of Gene Cloning: to make many copies/amplify particular gene and to produce a protein product from it
- Can isolate gene and give product to different organism
Using Restriction Enzymes to Make a Recombinant Plasmid
- Recombinant DNA tech uses Restriction enzymes (restriction endonucleases) to cut DNA
- Restriction enzymes obtained from bacteria that manufacture these enzymes to combat invading viruses
- DNA of a bacterial cell is protected from the cell’s own restriction enzymes by the addition of methyl groups (—CH3) within the sequences recognized by the enzymes
- Restriction enzymes cut the sugar phosphate backbones of the two DNA strands in a staggered manner at short, specific nucleotide sequences (restriction sites) → yielding a set of double-stranded restriction fragments with single-stranded sticky ends
- Sticky ends can form hydrogen-bonded base pairs with complementary sticky ends of a DNA molecules cut with the same restriction enzyme
- Restriction enzymes obtained from bacteria that manufacture these enzymes to combat invading viruses