Toolbox for a Genetic Engineer

1. Toolbox for a Genetic Engineer Goal: to make changes to an organisms DNA; to make recombinant DNA

2. Tool #1: Restriction enzymes • Early 1950’s Certain strains of E. coli bacteria were resistant to infections by bacterial viruses known as bacteriophages • Salvador Luria, GiusepeBertaini, and Jean Weigle • 1962 Discovery that E. coli bacteria that were resistant to infection by bacteriophage because they possess enzymes which recognize and destroy bacteriophage DNA • Werner and Arber: University of Geneva • Later named “restriction enzymes” also known as “RE’s” • 1968 Arber and group labeled bacteriophage DNA with radioactive isotope (32P) and inserted into E. coli known to possess restriction enzymes • Infected E. coli survived; analysis showed the radioactively labelled bacteriophage DNA was cut into many pieces • E. coli’s DNA was kept intact

3. Observations about RE’s from experimentation • When radioactively labeled bacteriophage was inserted into various strains of RE containing E. coli, the bacteriophage DNA was destroyed by being cut up into small pieces. • When placed in E. coli not containing RE’s, the bacteriophage replicated and killed the E.coli. • Bacteriophage DNA was more easily destroyed by E. coli strains that possessed two types of RE’s as compared to those possessing one type of RE • All strains of bacteriophages were not cleaved by E. coli known to possess RE’s • Suggested that RE’s cleaved (cut up) only bacteriophages containing specific sequences of DNA

4. RE’s: How do they work? • Highly specific • Scan DNA until a certain sequence of nucleotide bases is identified • Sequence is so specific it won’t happen at many sites • One, two or three sites • Restriction enzymes recognize a sequence such as: • 5′ G A A T T C 3′ Strand • 3′ C T T A A G 5′ Strand • RE can recognize the paired sequences from either strand • What do you notice about this sequence? • Read the same in either direction. • They are a palindrome. • The restriction enzyme can cut this palindromic sequence in one of two manners • across both strands at the same spot • in a staggered manner that yields free single-stranded ends called "sticky ends." These sticky ends have proved most useful in recombinant DNA work.

5. Names and Sources of RE’s • The names of restriction enzymes are derived from their bacterial sources • One of the enzymes most widely used in recombinant DNA work is EcoR1, which is isolated from Escherichia coli RY13 • Using EcoR1 on the above sequence, one would always obtain the ends: • ————– 5′ G     A A T T C 3′ ————– • ————– 3′ C T T A A      G 5′ ————– • Other examples include • HindII (isolated from Haemophilus influenza) • Xba I (isolated from Xanthomonasbadrii) • others • The specificity of each enzyme allows researchers to cut DNA in a predictable and reproducible manner. • Also called “restriction endonucleases”

7. Tool #2 Gel Electrophoresis • Lab technique used to separate pieces of • DNA • Parts of a protein (which would be?) • Technique separates molecules by • By charge • By size • For what purpose? • Isolated a specific sequence of DNA (for future research) • DNA fingerprinting

8. How does Gel Electrophoresis Work? • DNA is negatively-charged (why?) therefore, if loaded in the gel and positioned at the – (negative) pole it will move toward the positive electrode (+). • DNA fragments that are shortest will travel farthest • DNA fragments that are the longest will remain closest to the origin (travel a shorter distance. • Using the same basic principles, electrophoresis can also be used to separate • fragments of RNA. • proteins. • How would gel be set up differently for DNA or RNA compared to proteins?

9. Lab 6: Gel Electrophoresis • Conclusions DNA = negatively charged correlate distance to size smaller fragments travel faster & therefore farther

10. Tool #3: Plasmids • Small, circular DNA molecules found inside some bacteria cells • Self-replicating by using cell’s polymerases • Sometimes carry genes of their own • Especially useful are genes for antibiotic resistance • Tetracycline • Ampicillin • Kanamycin • Easy to extract (small) and purify from bacterial cells • When cut with an RE, they can take up foreign DNA (from any source-with “gene of interest”) which have been cut with the same RE. • Plasmid carrying “gene of interest” is now called a vector • Plasmid carrying “gene of interest” can now be taken up by a bacterial cell • When that bacterial cell replicates, what happens to the plasmid carrying the “gene of interest”? • How will we know the bacterial cell has that plasmid in it? • Screening procedure?

11. Tool #4 Ligase • Ligase enzyme is used to join the sugar-phosphate backbones of two DNA molecules • No matter their source