ADVANCES IN GENETICS RECOMBINANT DNA TECHNOLOGY Isolation of Genes: DNA is extracted using biochemical procedures from cells of an organism.

Once we have isolated DNA, we cut it at sites of specific nucleotide sequences by using enzymes called restriction endonucleases. These enzymes read the DNA and recognize specific sequences. When they find their specific sequence, they cut the DNA molecule at that site. How do you know you got the gene? After cutting up the DNA, you find out which piece does what by artificially expressing the gene, then looking for the gene product. You try to find the piece that codes for the protein that you want, whether that's a structural protein or an enzyme. That process is called in vitro translation. Moving Genes: Insertion into a prokaryote (bacterium). Isolate plasmids from bacteria. Plasmids are circular molecules of DNA that normally occur in bacteria. Cut open a plasmid using a restriction endonuclease and add the gene of your choice, and seal the whole thing back together using a ligase, an enzyme that will join DNA together. Put those plasmids back with living bacteria. The bacteria will take up the plasmids, multiply them so that you get many copies of the gene, which are of course multiplied again when the bacteria multiply, and you get lots of the product produced. Sometimes when you put a eukaryotic gene in a bacterium, the gene product is not made properly because of the presence of introns in the eukaryotic gene. To circumvent this problem, once can extract edited mRNA from the eukaryote and use reverse transcriptase (RNA-dependent DNA polymerase) to read mRNA and make a DNA copy of the mRNA. This produces an edited gene. We can then put that edited form into a bacterium and have it properly expressed. Insertion into a plant. Agrobacterium transformation - The bacterium Agrobacterium tumefaciens contains a plasmid called the Ti plasmid, which contains a stretch of DNA called T-DNA that gets copied and transferred into the plant's chromosomes. We can insert new genes in the T-DNA and let the bacterium deliver the gene into the plant for us. Protoplast Fusion Enzymatically digest away the cell wall, then cause the cell membranes to fuse and combine the protoplasm of the cells. The nuclei will fuse and the chromosomes will be merged to create a hybrid cell with merged characteristics. Insertion into a plant or animal cell. Viruses Have specific infectivity, but can only transfer very limited amounts of DNA.

Microprojectile Bombardment - Also called biolistic particle delivery, this technique uses small particles which are coated with DNA and propelled into the cell. The DNA is then taken up by the cell and joins into the chromosomes. Microinjection - direct injection of genes into nucleus of target cell. Electroporation - use of electricity to open transient pores in membrane of cell immersed in DNA solution. Cell takes in DNA. Liposomes - artificial lipid vesicles with DNA enclosed. Fuses with a cell and deposits new DNA into cell. Commercialization: Products on the Market Humulin = human insulin. Available now at relatively low cost because human insulin is now made by bacteria. Can isolate it from bacteria with greater ease and at lower cost than from human cadavers, where it used to be obtained. Interferons - used in treatment of cancer, currently working on other forms of interferon. Plasminogen-activator protein - Activates body's plasminogen which then dissolves blood clots obstructing coronary arteries. Prevents heart attacks. May be powerful enough to stop a heart attack in progress. Biopharmacology - the production of biologically active human chemicals in animals. For example, sheep have been engineered to produce antitrypsin in their milk. Antitrypsin is used to fight emphysema. Gene Therapy: Gene therapy is the transfer of a gene to a living organizm to correct a genetic deficiency. Trials are currently underway to transfer genes to humans to correct genetic disorders. Success has been obtained with delivery of ADA to fight SCIDS. Forensic Applications: RFLP analysis - Restriction Fragment Length Polymorphism Relies on the fact that we all have a lot of variation in our DNA, especially in the non-coding regions of our chromosomes. DNA is collected, fragmented by restriction enzymes, and the pieces are separated by size. This forms a genetic fingerprint or profile based on differences in fragment lengths. PCR - polymerase chain reaction This procedure uses DNA polymerase to repeatedly copy or amplify a DNA sample. It amplifies only DNA from specific regions of DNA. Then probes (radioactively labeled DNA sequences with homology for alleles of interest) are prepared and used to identify the presence or absence of certain alleles. If one screens for presence of certain alleles for as many as six different genes, one can generate a genetic indentity with highly significant probabilities.