2Genetic EngineeringGenetic engineering is the direct manipulation of genes for practical purposesManufacturing protein productsBiotechnology is the manipulation of organisms or their components to make useful productsSelective breeding of cattleMaking wine and cheeseGenetic engineering/biotechnology are major areas of research in biology today
3DNA Cloning Preparing identical copies of a specific gene Can be used to produce a protein product (gene protein)Copies of a gene can be used for:Basic researchEndowing an organism with a new metabolic capabilitypGLO
4Practical Applications of Gene Cloning Gene for pest resistance inserted into plantsGenes used to alter bacteria for cleaning up toxic wasteProtein dissolves blood clots in heart attack therapyHuman growth hormone treats stunted growthOthers??
5Restriction Enzymes & Recombinant DNA enzymes that cut DNA molecules at specific locationsLook for specific sequences:Ie: ATTACCGTARecombinant DNA –DNA in which nucleotide sequences from 2 different sources (often different species) are combined into the same DNA molecule
7Recombinant DNA Sticky Ends: Single-stranded ends of a restriction fragment (piece of DNA)Sticky ends of restriction fragments can form hydrogen-bonded base pairs with other sticky endsThese bonds are sealed with DNA ligase
8PlasmidsA plasmid is a DNA molecule that is not part of the chromosome in bacteriaOften give bacteria an “advantage” such as antibiotic resistancePlasmids are often used as cloning vectorsTake foreign DNA into a cell and replicate there
10Polymerase Chain Reaction (PCR) PCR is a process used to copy a segment of one or many DNA molecules quickly in vitro (in a test tube)PCR can make BILLIONS of copies of a target segment of DNA in a few hours
11Polymerase Chain Reaction (PCR) DenaturationMixture is heated to separate the DNA strands2. AnnealingMixture is cooled to allow primers to form (beginning of DNA replication)3. ExtensionDNA polymerase adds nucleotides to 3’ end of primer
13Applications of Polymerase Chain Reaction (PCR) Used to amplify (copy) DNA from a variety of sourcesFragments of DNA from a 40,000-year-old woolly mammothDNA from fingerprints or tiny amounts of blood, tissue, semen at crime scenes…If there is not much DNA available for analysis, PCR can produce enough DNA for other applications (electrophoresis, etc.)
14Restriction Fragment Analysis Restriction fragment analysis detects certain differences in the nucleotide sequences of DNA molecules
15Restriction Fragment Length Polymorphisms (RFLPs) RFLPs are differences in restriction sites on homologous chromosomes that result in different restriction fragment patternsEveryone has a different DNA sequence, so the restriction enzymes will cut the DNA at different placesThe length of the restriction fragments can be analyzed using electrophoresis
16Gel ElectrophoresisThe DNA fragments produced by restriction enzymes are sorted by gel electrophoresisSorted by rate of movement through a gel in an electric fieldDistance is inversely proportional to the length of the fragment –Long fragments don’t go far, short ones doThe banding pattern (where the fragments stop) is used to analyze/identify DNA
18The Human Genome Project Largely completed in 2003Essentially, a detailed map of the entire human genomeGenetic mapping –Relative order of genes (1st, 2nd, 3rd, etc.)Physical mapping –Definitive distance between genes (2457 bp, etc.)DNA sequencing –complete nucleotide sequence (ATCCG…)
19Applications of DNA Technology Medical ApplicationsDiagnosing genetic diseasesAmplifying (cloning) genes and analyzing themGene therapyAltering the genes of an afflicted individualReplacing a “defective” allele with a “normal” alleleTo be effective, it must be done in cells that multiply throughout lifeBone marrow cells (somatic cells)Gametic cells (sperm/egg)Lots of ethical issues involved here!
20Applications of DNA Technology Pharmaceutical ProductsUsing DNA cloning, genes that code for the production of specific proteins can be made in vitroHuman insulin (diabetes)Human growth hormone (HGH)Vaccines
21Forensic EvidenceDNA fingerprinting (RFLP analysis and electrophoresis) is used to solve crimesThe likelihood that 2 people would have the same DNA fingerprint (restriction enzymes would cut their DNA at all the exact same places and therefore have the same banding pattern) is between 1 in 100,000 and 1 in 1,000,000,000Also used in paternity testing
22Applications of DNA Technology Environmental CleanupMany bacteria can extract heavy metals (copper, lead, nickel) from their environmentsThese genes could be added to other organisms to clean up mining wastesBacterial strains can degrade some of the chemicals released during oil spills
23Ethics & DNA Technology The progress in genetic technology has raised new ethical questions:Who should have the right to examine someone else’s genes?How should that information be used?Should a person’s genome be a factor in suitability for a job or eligibility for insurance?