Recombinant DNA Methods: from Insulin to GMO's

Cloning Vector

3-4 features of every cloning vector *1) There must be a bacterial origin of replication. Replication can't start just anywhere. It always has to start at the origin of replication. 2) Antibiotic resistance gene: allows us to specifically to select agains the bacteria that do NOT have our plasmid of interest 3) Need a promotor 4) Need a multi-cloning site Plasmids, bacteriophages, yeast artificial chromosomes

Palindromic restriction sites

Allow restriction enzymes to cut strand-independently. GAATTC - no matter which direction we are reading it from, the enzyme will cut it. GAATTC vs. CTTAAG

3 Classes of restriction enzymes

Blunt ends - when ends are even Sticky ends - when ends are uneven 5' overhang and then the 3' overhang Sticking together blunt ends is not as efficient, easy, or stable. Need high enough concentration for ligase to stabilize them. You can stick together a blunt end with a staggered end (5' overhang) by polymerase - fill in overhangs. Generate a blunt end from the sticky end first, and then ligate them together with another blunt end. Can't fill in 3' overhangs because replication only works in one direction. You could use an enzyme to chew the overhang off, but you certainly couldn't fill it.

3 Basic steps of the PCR chain

Denature ~95 degrees celsius Anneal ~55-70 degrees celsius Elongate ~72 degrees celsius Exponential DNA amplification by PCR: first cycle = 2 double stranded second cycle = producing 4 double stranded third cycle = producing 8 double stranded

Uses of Restriction Enzymes

Determining the organization of genome, physical map of DNA: first use was to show that SV40 DNA was circular and determine the position of the origin of replication Distinguishing alternative alleles within the human genome: restriction fragment length polymorphisms (RFLPs) Molecular cloning (producing recombinant DNA molecules) isolate and amplify DNA for analysis of genes

Genomic Library Screening

For PCR, you need to already know the sequence. Screen bacteria for phenotype of interest in genomic library. Look at diagram

Genetic Engineering vs. Cloning

Genetic engineering = directly modifying an organism's genome. Once an organism is engineered, it is often propagated by cloning Cloning = producing genetically identical individuals. Ex: supermarket bananas are cloned but no genetically altered.

Site-directed mutagenesis

Insulin that is manufactured is stabilized, ensured that it will not cross-react with the immune system, and has other properties. (5') GATCGATCGATC (3') (3') CTAGCTCGCTAG (5') ^we don't need to have a perfectly matched Oligo (Its another name for the primers that are used for PCR) When we isolate all these plasmids, we get two populations that we need to differentiate.

Case Study: Insulin and rDNA

Normally you take in sugar and release insulin from your pancreas, which helps the body reduce blood sugar. Diabetes is a condition in which the body attacks its own cells that produce insulin.

Amplifying the insulin gene for cloning

PCR - insulin gene - subcloning - bacterial insulin expression plasmid - transformation and expression - e. coli expressing insulin = verification Use PCR to amplify insulin gene... PCR generates a linear piece of DNA. We don't wan't the insulin gene, we want the insulin protein. To do this, we need to transcribe and translate the gene to protein. Cells are really good at this, so we are going to take our gene and put it into a different cell.

Cloning Vector

Preparation: digestion with restriction enzyme Isolation: of DNA to insert into vector Litigation: of DNA fragment to vector Mix our linear plasmid with the PCR product and we put them together. In order to make sure the green doesn't attach to itself, we make sure to have an excess of blue.

Primer design and hybridization

Primer with attached restriction enzyme site. Moves 3' to 5'

Transformation of E. Coli

Recombinant DNA - introduced into bacterial cell, some cells take up the plasmid, cell culture produces hundreds of millions of new bacteria, many copies of purified plasmid isolated from lysed bacteria. Take our cells, shock them to open up their pores to allow DNA in more efficiently, small fraction of bacteria will take plasmid of interest. Must get rid of untransformed bacteria to stop them from competing with transformed bacteria - have transformed bacteria be resistant to an antibiotic in the agar. The recombinant clones, transformed will not die when antibiotics administered while the other original bacteria will die. Therefore, The bacteria growing on the agar gel will be all the plasmid-containing DNA.

Discovery of Insulin

Remove the dog's pancreas, the dog develops diabetes. Take the islets of Langerhans isolated from the pancreas - the cells liquidized and filtered off - inject the solution back into the dog and the dog's diabetes is treated successfully. Ground up to get an extract, and fractionate out the parts to determine which part was the functioning part by injecting different parts into the dog.

Restriction Enzymes

Restriction Endonucleases Created to chew up foreign DNA. Primitive form of immune system for bacteria - typically methylate their DNA, a way to mark DNA as their own. When foreign DNA comes in from viruses or plasmids, the bacteria can recognize this DNA as foreign (since it isn't methylated) and chop it up. Some cut at specific sequences... shorter sequences occur more frequently. Nonspecific binding of Enzyme -- sliding/hopping and jumping to the specific binding, coupling = the conformational change in enzyme. DNA activates the catalytic center. Catalysis (requires Mg2+) which causes a kink in the DNA, breaks the phosphodiester bond, which releases the product.

Cut and Paste

Restriction Enzyme Digestion and Litigation of cut DNA Use DNA ligase for ligation - joining of cohesive ends, annealing of fragments, and then ligation with DNA ligase (catalyzes formation of phosphodiester bond).

Golden Rice

Rice lacks 2 enzymes needed for making the vitamin A precursor beta-carotene. Golden rice is engineered by adding two genes from daffodil and soil bacteria that allow rice to produce beta-carotene, a precursor of vitamin A

Sanger sequencing

Sanger "deoxy" sequencing: takes advantage of the chemistry of the nucleotides. Without the 3' OH, it can still be added to the chain, but it cannot be further extended. "Terminators." We want to add a lot of normal nucleotides, but add in a small proportion of dideoxy in order to terminate. Look at color

Expression Cloning of GFP

Screen bacteria for phenotype of interest - the dark colonies have the vector but not the gfp.

Directional Cloning

Two different enzymes: To deal with this, we could sort out the wrong bits, OR, we could use a directional cloning enzyme. We cut our DNA with TWO different enzymes, so that we get 2 benefits: 1) the green won't join to itself and 2) the blue won't go backwards.

Symmetric Cloning

Two orientations for inserted DNA - sticky ends are going to be the same, so our gene of interest has a 50/50 chance of being inserted backwards.

Bacteria

carry extrachromosomal plasmids - can't sustain life themselves, piggyback on the reproductive process - as well as chromosomes.

Pros of GMOs (genetically engineering in plants)

herbicide resistant crops insect resistant crops rice with vitamin A slow ripening fruit-papaya controlled crop ripening-coffee beans virus resistant potatoes

Potential Cons of GMOs

nutritional concerns? - largely unsupported to date (increased toxicity/allergenicity, decreased nutrients) Increased spread of antibiotic resistance Contaminating wild species (environmental risk)

Plasmids

self-replication, contain origin of replication. Can be cut by restriction enzymes and sealed by ligase. Circular double stranded DNA (cloning vector) --> cleaved by restriction nuclease. The DNA fragment to be cloned is covalently linked with DNA ligase --> recombinant DNA Recombinant engineering: cut the circular plasmid, put the DNA of interest inside, and seal it up. Now the plasmid has our gene of interest.

Agarose gels and ethidium bromide

we use ethidium bromide, which evenly inter locates in the DNA. It is fluorescent, so we can see glowing bands.

Insulin

wildly successful - purified extract given to comatose children with diabetes. But it is very hard to obtain. 1920s and 60-70 years on, insulin was taken from animal pancreases, and purified... but only a tiny amount could be taken. Usually isolated from calf or pig pancreas. Beta cells inside the islets of Langerhans... blood vessel and acinus.