frontiers of biotechnology
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Frontiers of Biotechnology. Manipulating DNA. Scientists use several techniques to manipulate DNA. Restriction Enzymes cut DNA. Why cut DNA? To study specific genes instead of ALL the genes on a chromosome Restriction enzymes act as molecular scissors Recognize specific sequences - PowerPoint PPT PresentationTRANSCRIPT
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FRONTIERS OF BIOTECHNOLOGY
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MANIPULATING DNA• Scientists use several techniques to
manipulate DNA
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RESTRICTION ENZYMES CUT DNA• Why cut DNA?– To study specific genes
instead of ALL the genes on a chromosome
• Restriction enzymes act as molecular scissors– Recognize specific
sequences• Some leave “blunt ends”• Some leave “sticky ends”
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RESTRICTION MAPS SHOW THE LENGTHS OF DNA FRAGMENTS
• Gel Electrophoresis: a technique that uses an electrical field within a gel to separate molecules by their size
• DNA is negatively charged and moves toward the positive pole when the electrical field is applied
• Smallest DNA fragments move the fastest
• A pattern of bands is formed
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GEL ELECTROPHORESIS
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POLYMERASE CHAIN REACTION• PCR: technique
that produces millions of copies of a specific DNA sequence in just a few hours
• Invented by Kary Mullis in 1983
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PCR• Uses: – DNA to be copied– DNA polymerase– Plenty of nucleotides A, T, C, and G– Two primers
• 3 Step Process:– Separating– Binding– Copying
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RFLPs• Restriction Fragment Length Polymorphisms
• No two individuals have the same genetic material except identical twins
• Restriction enzymes cut at different places, depending on the DNA sequence
• The lengths of DNA restriction fragments are different between two individuals
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DNA FINGERPRINTING• A DNA fingerprint is a type of
restriction map
• Representation of parts of a individual’s DNA that can be used to identify a person at the molecular level
• Focuses on noncoding regions of DNA, or DNA sequences outside genes
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DNA FINGERPRINTING• DNA sample from:
– Blood– Semen– Bone – Hair
• …Useful in forensics!
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DNA FINGERPRINTING IS USED FOR IDENTIFICATION
• DNA fingerprints and probability– Compare at least 5 regions of the
genome
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GENETIC ENGINEERING• Entire organisms can be cloned
• Clone: genetically identical copy of a gene or of an organism
• New genes can be added to an organism’s DNA
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4 BASIC STEPS TO GENETIC ENGINEERING
• 1. Cutting DNA
• 2. Making recombinant DNA
• 3. Cloning
• 4. Screening
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STEP 1: CUTTING DNA
• The DNA from the original organism containing the gene of interest is cut by restriction enzymes
• Restriction Enzymes: bacterial enzymes that destroys foreign DNA molecules by cutting them at specific sites
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STEP 1: CUTTING DNA
• Vector: Any agent, such as a plasmid, that carries the gene of interest into another cell
• Plasmid: A circular DNA molecule that is usually found in bacteria and that can replicate independent of the main chromosome
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RECOMBINANT DNA
• DNA molecules that are artificially created
• HOW?????
• Created by combining DNA from different sources
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EXAMPLE: INSULIN• A protein hormone that controls sugar
metabolism
• Diabetics cannot produce enough
• Must take doses of insulin daily
• Before genetic engineering, insulin was extracted from the pancreases of slaughtered cows and pigs and then purified
• Today the human insulin gene is transferred to bacteria through genetic engineering
• Because the genetic code is universal, bacteria can transcribe and translate the human insulin gene
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STEP 2: MAKING RECOMBINANT DNA
• DNA fragments from the gene of interest are combined with the DNA fragments from the vector
• DNA ligase: an enzyme that bonds the DNA fragments together
• The host cell then takes up the recombinant DNA
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STEP 3: CLONING
• Gene Cloning: many copies of the gene of interest are made each time the host cell reproduces
• Remember: bacteria reproduce by binary fission, producing identical offspring with the plasmid DNA!
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STEP 4: SCREENING• Cells that have received the particular gene
are separated from the cells that did not take up the vector with the gene of interest
• The cells can transcribe and translate the gene of interest to make the protein coded for the gene
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CONFIRMATION OF A CLONED GENE
• Southern Blot: a technique used to test for the presence of a specific gene
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NORTHERN BLOT• Similar to a
Southern Blot
• Uses RNA instead of DNA
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GENETIC ENGINEERING PRODUCES ORGANISMS WITH
NEW TRAITS
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SELECTIVE BREEDING• Allowing only those animals with
desired characteristics to produce the next generation
• Horses, cats, farm animals, crops
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HYBRIDIZATION
• Crossing dissimilar individuals to bring together the best of both organisms
• Hybrids: the individuals produced from such crosses
• For example, a disease resistant plant and the food producing capacity of another
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INBREEDING• The continued breeding of individuals with
similar characteristics
• Often seen in dogs
• Retains characteristics but has risks
• Genetically similar individuals could bring together two recessive alleles for a genetic defect
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TODAY…GENETIC ENGINEERING
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GENETICALLY ENGINEERED CROPS• More tolerant to
drought
• Plants that can adapt to different soils, climates, and environmental stresses
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GENETICALLY ENGINEERED CROPS• Resistant to
biodegradable weedkiller Glyphosate (kills weeds but now doesn’t kill the crop)
• Resistant to insects (gene injures the gut of chewing insects)-therefore plant doesn’t need to be sprayed with pesticides
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MORE NUTRITIOUS CROPS• Improve the nutritious value
of many crops
• Asia: rice is a staple food
• Low in iron an beta carotene
• Iron deficient and poor vision
• Genetic engineers have added genes to rice from other plants to overcome this deficiency
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POTENTIAL PROBLEMS TO GM CROPS
• Concern that some weeds will become resistant to the weed killer Glyphosate
• New weed-control alternatives will have to be implemented
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POTENTIAL PROBLEMS TO GM CROPS
• Nutritional value has been increased in many crops
• Crops must be tested to make sure consumers are not allergic to the GM product
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GENE TECHNOLOGY: ANIMAL FARMING
• Farmers added growth hormones to the diet of cows to increase milk production
• Growth hormone was extracted from the brains of dead cows
• The hormone was introduced into bacteria and added as a supplement to a cow’s diet
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TRANSGENIC ANIMALS• Animals that have
foreign DNA in their cells
• Human genes have been added to farm animals in order to get the farm animals to produce human proteins in their milk
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TRANSGENIC ANIMALS• This is complex and cannot be made by
bacteria through gene technology
• Human proteins are extracted from the animal’s milk and sold for pharmaceutical purposes
• Cloning animals: creating herds of identical animals that can make medically useful proteins
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CLONING FROM ADULT ANIMALS• The intact nucleus of an embryonic or fetal
cell (whose DNA has been recombined with a human gene) is placed into an egg whose nucleus has been removed
• The egg with the new nucleus is put in the uterus of a surrogate, or substitute mother and allowed to develop
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CLONING FROM ADULT ANIMALS
• 1997 Ian Wilmut first successful cloning using differentiated cells from an adult animal
• Dolly the sheep
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CLONING FROM ADULT ANIMALS• Differentiated cells: cells that
have become specialized to become specific cell types
• Scientists had thought that embryonic or fetal cells were the only way…wrong!
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CLONING FROM ADULT ANIMALS• Mammary cells from one sheep were fused
with egg cells without nuclei form a different sheep
• The fused cells divided to form embryos, implanted into surrogate mothers
• Only one survived the cloning process
• Dolly, identical to the sheep that provided the mammary cell
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PROBLEMS WITH CLONING
• Only a few of the cloned offspring survive for long
• Many become fatally oversized
• Problems in development
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GENOMIC IMPRINTING• The right combination of genes are
turned “on” and “off” during early development
• The egg takes years to develop the genomic imprint
• In cloning, the egg divides within minutes
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GENOMIC IMPRINTING
• Reprogramming is not possible in such a short time
• Critical errors in development can occur
• Because of these technical problems and ethical problems, cloning humans is illegal in most countries
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CONCERNS ABOUT GENETIC ENGINEERING
• Ethical?
• GM crops
– Not enough research had been done to see if added genes might cause allergic reactions or have other unknown side effects
– Interbreeding with natural plants…what does it mean?
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GENOMICS INVOLVES THE STUDY OF GENES, GENE FUNCTIONS, AND ENTIRE
GENOMES
• Genomics: The study of genomes, which can include the sequencing of all of an organism’s DNA
• Gene sequencing: determining the order of DNA nucleotides in genes or in genomes
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THE GEOGRAPHY OF THE GENOME
• Only 1-1.5% of the human genome codes for proteins
• Each human cell contains about 6 feet of DNA
• Less than 1 inch are exons
•
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THE GEOGRAPHY OF THE GENOME
• Human cells contain about 25,000 genes (scientists had expected 120,000!)
• Only 2x the number of genes in a fruit fly!
• Many human genes are identical to those of other species
• All humans are genetically close (DNA of any 2 people is 99.9% identical)
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THE HUMAN GENOME PROJECT• Our genome is relatively small! 3 billion base
pairs, but only between 30,000-40,000 genes
• Project started in 1990 with 2 main goals:
– Map and sequence all of the DNA base pairs of the human chromosomes (accomplished in 2003)
– Identify all of the genes within the sequence (still be worked on)
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THE HUMAN MICROBIOME PROJECT• 200 scientists at 80 institutions
sequenced the genetic material of bacteria taken from nearly 250 healthy people
• As many as a thousand bacterial strains on each person.
• Each person’s collection of microbes, the “microbiome”, was unique
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TECHNOLOGY ALLOWS THE STUDY AND COMPARISON OF BOTH GENES AND
PROTEINS• Bioinformatics: the use of computer databases
to organize and analyze biological data
• DNA microarrays: tools that allow scientists to study many genes, and their expression at once; a small chip dotted with the genes being studied
• Proteomics: the study and comparison of all the proteins that result from an organism’s genome
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GENETIC SCREENING AND GENE THERAPY
• Genetic screening: the process of testing DNA to determine a person’s risk of having or passing on a genetic disorder
• Gene therapy: the replacement of a defective of missing gene, or the addition of a new gene, into a person’s genome to treat a gene
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GENETICALLY ENGINEERED DRUGS & VACCINES
• Possibilities for the applications of genetic engineering are endless!
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DRUGS• Many genetic disorders and other human
illnesses occur when the body fails to make critical proteins
• Example: juvenile diabetes– Body is unable to control levels of sugar in the blood
because the protein insulin cannot be made
• Example: Hemophilia– Factor VIII, a protein that promotes blood clotting– Donated blood was sometimes infected with HIV
and hepatitis B– Genetically engineered factor VIII eliminates these
risks
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VACCINESTraditional Vaccines
• Many viral diseases, such as smallpox and polio, cannot be treated effectively by existing drugs
• Vaccine: a solution containing all or part of a harmless version of a pathogen (disease-causing microorganism)
• When the vaccine is injected, the immune system recognizes the pathogen’s surface proteins and responds by making defensive proteins called antibodies
Genetically Engineered Vaccines• Avoid the danger of giving a
patient a disease
• The genes that encode the pathogen’s surface proteins can be inserted into the DNA of harmless viruses, such as cowpox
• The modified, harmless cowpox virus becomes an effective, safe vaccine