Ap Biology Chapter 20 Notes
Chapter 20: DNA Technology
Biotechnology: Use of living organisms to perform tasks. * Wine & cheese * Selective breeding * Antibiotic production * Recombinant DNA
Restriction Enzymes * Bacterial enzymes: cut up foreign DNA * Specific: only but at recognition sequences * Palindromic: cut at the same base sequence on each strand, but in the opposite direction * The exposed bases provide “sticky ends” * H-bond to compliment bases of segments cut with same restriction enzyme
Recombinant DNA
1. Isolate DNA from 2 sources * Bacterial plasmid w/2 genes * ampR(resistance to ampicillin) * lacZ(makes β galactosidase) * Human cell
2. Treat both w/ the same restriction enzyme * Recognition sequence cuts through lacZ gene in plasmid
3. Mix all DNA fragments with plasmids. * H-bonds bt/n bases temporarily hold “sticky ends” together
4. Add DNA ligase: creates permanent covalent bonds
5. Reintroduce plasmid to bacteria via transformation
6. I.D. recombinant cells by * Ability to grow in * Inability to digest x-gal(the colonies will be white, not blue)
X-gal -------------------------------- (β galactosidase)---------------------> Blue product
(molecular mimic of (white)lactose) lacZ gene
7. Each bacterium replicates to form thousands of clones * Many different human DNA fragments have been cloned * -------------------------------------------------
How do you find the one “of interest”?
Nucleic Acid Hybridization * Synthesize a Nucleic Acid Probe * Single strand DNA or RNA (radioactive) * Complementary to the gene of interest * Synthesize on the base of: * Base sequence of the gene (at least part of it) * AA sequence of the protein * Denature the DNA with heat or chemicals * The probe will H-bond to the appropriate gene
Genomic Library * Complete set of recombinant clones (includes all fragments) * Stored in a collection of phage clones (virus)
Biotechnology: Use of living organisms to perform tasks. * Wine & cheese * Selective breeding * Antibiotic production * Recombinant DNA
Restriction Enzymes * Bacterial enzymes: cut up foreign DNA * Specific: only but at recognition sequences * Palindromic: cut at the same base sequence on each strand, but in the opposite direction * The exposed bases provide “sticky ends” * H-bond to compliment bases of segments cut with same restriction enzyme
Recombinant DNA
1. Isolate DNA from 2 sources * Bacterial plasmid w/2 genes * ampR(resistance to ampicillin) * lacZ(makes β galactosidase) * Human cell
2. Treat both w/ the same restriction enzyme * Recognition sequence cuts through lacZ gene in plasmid
3. Mix all DNA fragments with plasmids. * H-bonds bt/n bases temporarily hold “sticky ends” together
4. Add DNA ligase: creates permanent covalent bonds
5. Reintroduce plasmid to bacteria via transformation
6. I.D. recombinant cells by * Ability to grow in * Inability to digest x-gal(the colonies will be white, not blue)
X-gal -------------------------------- (β galactosidase)---------------------> Blue product
(molecular mimic of (white)lactose) lacZ gene
7. Each bacterium replicates to form thousands of clones * Many different human DNA fragments have been cloned * -------------------------------------------------
How do you find the one “of interest”?
Nucleic Acid Hybridization * Synthesize a Nucleic Acid Probe * Single strand DNA or RNA (radioactive) * Complementary to the gene of interest * Synthesize on the base of: * Base sequence of the gene (at least part of it) * AA sequence of the protein * Denature the DNA with heat or chemicals * The probe will H-bond to the appropriate gene
Genomic Library * Complete set of recombinant clones (includes all fragments) * Stored in a collection of phage clones (virus)