DNA Technology

1. DNA Technology • Biotechnology is the process of manipulating organisms or their components for the purpose of making useful products. • Genetic engineering is the process of manipulating genes and genomes. • Recombinant DNA is DNA that has been artificially made, using DNA from different sources and from different species. An example is the introduction a human gene into an E. coli bacterium. • Gene cloning is the process by which scientists can produce significant samples of specific genes. • Polymerase chain reaction (PCR) is a method used to greatly amplify a piece of DNA without the use of cells – makes lots of copies of DNA. Can be used for all organisms and viruses.

2. Restriction Enzymes • Restriction enzymes are used to cut strands of DNA at specific sites (called restriction sites) & make genetic engineering possible • When a DNA molecule is cut by restriction enzymes, the result will always be a set of restriction fragments that will always have at least one single-stranded end called a sticky end. Sticky ends can form hydrogen bonds with complementary single-stranded pieces of DNA (sealed with DNA ligase). • Analyzing the restriction fragments allows detection of differences in DNA that affect the location and number of restriction sites. • Gel electrophoresis is a technique that separates macromolecules on the basis of their size and charge with the use of an electrical current.

3. 3 1 2 Recombinant DNA Restriction site 5 3 G A A T T C DNA 5 3 C T T A A G Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow G A A T T C C T T A A G Sticky end A A T T C G G DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. C T T A A Fragment from differentDNA molecule cut by thesame restriction enzyme G A A T T C A A T T C G C T T A A T T A A C G G One possible combination DNA ligaseseals the strands. Recombinant DNA molecule

4. 1 2 Mixture of DNA molecules of differ- ent sizes Cathode APPLICATION TECHNIQUE RESULTS Gel Power source Glassplates When the current is turned on, the negatively charged DNA molecules move toward the positive electrode, with shorter molecules moving faster than longer ones. Bands are shown here in blue, but on an actual gel, DNA bands are not visible until a DNA-binding dye is added. The shortest molecules, having traveled farthest, end up in bands at the bottom of the gel. Anode Longermolecules Shortermolecules Gel Electrophoresis Gel electrophoresis is used for separating nucleic acids or proteins that differ in size, electrical charge, or other physical properties. DNA molecules are separated by gel electrophoresis in restriction fragment analysis of both cloned genes (see Figure 20.9) and genomic DNA (see Figure 20.10). Each sample, a mixture of DNA molecules, is placed in a separate well near one end of a thin slab of gel. The gel is supported by glass plates, bathed in an aqueous solution, and has electrodes attached to each end. Gel electrophoresis separates macromolecules on the basis of their rate of movement through a gel in an electric field. How far a DNA molecule travels while the current is on is inversely proportional to its length. A mixture of DNA molecules, usually fragments produced by restriction enzyme digestion, is separated into “bands”; each band contains thousands of molecules of the same length. After the current is turned off, a DNA-binding dye is added. This dye fluoresces pink in ultraviolet light, revealing the separated bands to which it binds. In this actual gel, the pink bands correspond to DNA fragments of different lengths separated by electrophoresis. If all the samples were initially cut with the same restriction enzyme, then the different band patterns indicate that they came from different sources.

5. Normal  -globin allele 201 bp Large fragment 175 bp DdeI DdeI DdeI DdeI Sickle-cell mutant -globin allele Large fragment 376 bp Ddel Ddel Ddel (a) DdeIrestriction sites in normal and sickle-cell alleles of -globin gene. Sickle-cellallele Normalallele Largefragment 376 bp 201 bp175 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles. Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the -globin gene

6. Disease diagnosis: if the sequence of a particular virus’s DNA is known, PCR can be used to amplify a patient’s blood sample to detect even small traces of the virus Gene therapy: the alteration of a individual’s genes, great potential in treating disorders traced to a single defective gene Pharmaceutical production: large amounts of protein can be produced (e.g., human insulin) Forensics: DNA samples from blood, skin or hair of alleged criminals can be compared to crime scene DNA. Paternity: DNA fingerprinting can also be used to determine paternity Environmental clean up: scientists engineer microbes with a fondness for pollutants Applications of DNA Technology

7. Blood from defendant’s clothes Victim’s blood (V) Defendant’s blood (D) 4 g 8 g V Jeans D shirt DNA fingerprints from a murder case

8. DNA Transformation • The uptake of foreign DNA • Bacterial cells can take up small pieces of circular DNA called plasmids that have been engineered with specific genes • One plasmid, pAMP, contains a gene for ampicillin resistance. • Normally, E. Coli cells are destroyed by the antibiotic ampicillin, but E. Coli cells that have been transformed by taking up the pAMP plasmid will be able to grow on agar plates containing ampicillin. • Thus, we can select for transformants • Those cells that are not transformed will be killed by ampicillin • Those that have have been transformed will survive. • Organisms other than bacteria can take up foreign DNA as well and are called transgenic

9. 2 1 3 Agrobacterium tumefaciens Genes conferring useful traits, such as pest resistance, herbicide resistance, delayed ripening, and increased nutritional value, can be transferred from one plant variety or species to another using the Ti plasmid as a vector. APPLICATION TECHNIQUE RESULT Tiplasmid Site where restriction enzyme cuts The Ti plasmid is isolated from the bacterium Agrobacterium tumefaciens. The segment of the plasmid that integrates into the genome of host cells is called T DNA. T DNA DNA with the gene of interest Recombinant Ti plasmid Isolated plasmids and foreign DNA containing a gene of interest are incubated with a restriction enzyme that cuts in the middle of T DNA. After base pairing occurs between the sticky ends of the plasmids and foreign DNA fragments, DNA ligase is added. Some of the resulting stable recombinant plasmids contain the gene of interest. Recombinant plasmids can be introduced into cultured plant cells by electroporation. Or plasmids can be returned to Agrobacterium, which is then applied as a liquid suspension to the leaves of susceptible plants, infecting them. Once a plasmid is taken into a plant cell, its T DNA integrates into the cell‘s chromosomal DNA. Transformed cells carrying the transgene of interest can regenerate complete plants that exhibit the new trait conferred by the transgene. Plant with new trait Transgenic Plants