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DNA Technology. Chapter 13 Lab Biology 1A Chapter 15 Lab biology 2b Chapter 13 Honors Biology 4A. DNA Technology. Technology used to help with genetic engineering helps us : 1. identify genes for specific traits 2. transfer genes for a specific trait from one organism to another
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DNA Technology Chapter 13 Lab Biology 1A Chapter 15 Lab biology 2b Chapter 13 Honors Biology 4A
DNA Technology • Technology used to help with genetic engineering helps us: • 1. identify genes for specific traits • 2. transfer genes for a specific trait from one organism to another • 3. cure disease • 4. treat genetic disorders • 5. improve food crops
How can you get a desired trait without directly manipulating the organisms DNA? • HYBRIDIZATION; crossing organisms of different traits to produce a hardier product ex: mule • INBREEDING/SELECTIVE BREEDING; maintain the present genes by breeding only within the population ex: pedigree animals • INDUCING MUTATIONS; radiation, chemicals polyploidy (3N or 4N) plants resulted larger and hardier
Now let us manipulate the genes by altering the organisms DNA • DNA Technology: sci. involved in the ability to manipulate genes/DNA • Cure disease • treat genetic disorders • Improve crops
Tools: • DNA extraction • Restriction enzymes • Gel electrophoresis • DNA ligase • Polymerase chain rxn. (PCR)
Method: (5 steps) • Extract gene insulin • Cut insulin producing gene out using “restriction enzymes” • Sticky ends create overhang • Blunts no overhangs • Cutting clone vector cut plasmid with same restriction enzyme • Ligation: donor gene is spliced into plasmid DNA, DNA ligase glues it (this forms recombinant DNA = plasmid DNA + new piece of DNA) • Plasmid returned to bacterium & reproduces using donor gene in it (this is transgenic organism = organism with foreign DNA incorporated in it’s genome) • *reproduce*
Restriction Enzymes • BACTERIAL ENZYMES are used to cut DNA molecule into more manageable pieces • They recognize certain sequences • Creating “single-chain” tails in DNA called STICKY ENDS
Sticky Ends • Readily bind to complimentary chains of DNA therefore pieces of DNA that have been cut with the same restriction enzyme can bind together to form a new sequence of nucleotides • Recognizes CTTAAG
Cloning Vectors • Cloning vector is a carrier that is used to clone a gene and transfer it from one organism to another. • Many bacteria contain a cloning vector called a PLASMID. • PLASMID is a ring of DNA found in a bacterium in addition to its main chromosome.
PROCEDURE • To be used as a cloning vector in gene transfer experiments a plasmid is isolated from a bacterium. • Using restriction enzymes the plasmid is then cut and a DONOR GENE (specific gene isolated from another organism is spliced into it) • Then the plasmid is returned to the bacterium, where it is replicated as the bacterium divides, making copies of the donor gene. • Plasmid now contains a GENE CLONE
Cloning Vectors • !
Plasmid • *
Transplanting genes • In some cases, plasmids are used to transfer a gene to bacteria so that the bacteria will produce a specific protein • Ex: INSULIN = protein that controls sugar metabolism • Bacteria that receives the gene for insulin will produce insulin as long as the gene is not turned off
Steps: • 1. ISOLATING A GENE – isolate the DNA from human cells and plasmids from the bacteria • Use restriction enzyme • Splice human DNA into plasmids to create a genomic library (set of thousands of DNA pieces from a genome that have been inserted into a cloning vector)
Steps cont… • 2. PRODUCING RECOMBINANT DNA = combination of DNA from 2 or more sources • Inserting a donor gene such as human gene for insulin, into a cloning vector, such as bacterial plasmid results in a recombinant DNA molecule!
Steps cont… • 3. CLONING DNA – the plasmid containing recomb. DNA is inserted into a host bacterium (called transgenic organism • The trans. Bact. Is placed in a nutrient medium where it can grow and reproduce.
Expression of Cloned Genes • Sometimes PROMOTERS must also be transferred so the genes will be turned on. • Genes are often turned off until the proteins they code for are needed.
Practical uses of DNA Technology • Pharmaceutical products: insulin, HBCF (human blood clotting factor) • Genetically engineered vaccines • Increased agriculteral yields • Improving quality of produce • Slow down ripening • Enhance color • Reduce fuzz • Increase flavor • Frost resistance
Negatives • Allergies • Label’s don’t include all information • May create “super weeds”
Gene therapy • Treatment of genetic disorders • Ex: cystic fibrosis
DNA Technology Techniques • I. DNA Fingerprints pattern of bands made up of specific fragments from an individual’s DNA • USED FOR: • DETECTION OF A RELATIVE • SIMILARITIES BETWEEN SPECIES
How do you make DNA fingerprints? • RFLP (restriction fragment length polymorphism) analysis • 1. extract DNA from specimen using restriction enzymes • 2. separate fragments of DNA using electrophoresis (separates DNA according to size and charge) • 3. placed in wells made on gel • 4. electric current run through gel
Continue… • 5. negative fragments migrate to positive charged end of gel but not all at same rate • 6. pores in gel allow smaller fragments to migrate faster separating fragments by size. • 7. blotted onto filter paper.
Accuracy of DNA Fingerprints • DNA fingerprints are very accurate • However, genetic tests can only absolutely disprove, not prove, relationship! • Courts accept 99.5% accuracy as proof of alleged paternity
Polymerase Chain Reaction (PCR) • Used when you only have a TINY piece of DNA • PCR can be used to quickly make many copies of selected segments of the available DNA • Use a PRIMER to initiate replication • DNA doubles every 5 minutes
PCR is used for: • 1. crimes • 2. diagnosing genetic disorders from embryonic cells • 3. studying ancient fragments of DNA (tiny amounts)
HUMAN GENOME PROJECT • 2 GOALS: • 1. determine nucleotide sequence of entire human genome (aprox 3 billion nucleotide pairs or about 100,000 genes • 2. map the location of every gene on each chromosome
1996 • 1 % of 3 billion nucleotide pairs of DNA human genomes were analyzed • This allows for us to identify and determine the function of 16,000 genes!
Gene Therapy • Treating a genetic disorder by introducing a gene into a cell or by correcting a gene defect in a cell’s genome. • Ex: Cyctic fibrosis cause one defective gene malfunction of one protein
Gene Therapy for Cyctic Fibrosis • Nasal spray carrying normal cyctic fibrosis gene to cells in nose and lungs • Must repeat treatment periodically
Ethical Issues • Many people worry about how personal genetic information will be used: • Insurance??? • Employment???? • Human Genome Project will undoubtedly involve ethical decisions about how society should use the information! WHAT DO YOU THINK??
Practical Uses of DNA Technology • 1. produce perscription drugs • Vaccine (harmless version of a virus or a bacterium) • Pathogen (disease causing agent) treated chemically or physically so that they can no longer cause disease. • Pathogen (Ag) Antibody (Ab) • DNA tech. may produce vaccines safer than traditional ones!
Increasing Agricultural Yields • DNA Tech. used to develop new strains of plants • Ex: scientists can make tomato plants toxic to hornworms and effectively protect the plant from these pests.
Crops that do NOT need fertilizer • Plants require NITROGEN to make proteins and nucleic acids • Most plants get their N from the soil • TRANSGENIC FOOD CROPS contain genes for nitrogen fixation so they can grow in nitrogen POOR soil.
Genetically Engineered Foods • Foods may have toxic proteins or substances causing ALLERGIES • Ex: changing the gene that codes for an enzyme to ripening in tomatoes they are able to make tomatoes ripen without becoming SOFT!!
Genetically Engineered Crops • Some are concerned that genetically engineered crops could spread into the wild and wipe out native plant species. • SUPERWEEDS!!!!!!!!