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Genetic Modification and BiotechnologyTopic 3.5Manipulating the Genome involves cleaving DNA into small fragments and inserting the fragments into another organism.Used in medicine, agriculture, industry, research

Genetic EngineeringBig Questions:What is the relationship between science and technology?What are the implications of genetic technologies for society?What ethical and moral responsibilities do scientists have?Restriction EnzymesGene cloning and genetic engineering were made possible by the discovery of restriction endonucleases that cut DNA molecules at specific locations.

Restriction EnzymesIn nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria.DNA scissorsHundreds have been isolated and named after bacteria they were isolated from

Restriction EnzymesRestriction enzymes cleave [cut] both strands of the DNA molecule at certain points called palindromes.

"Was it a rat I saw?" "Step on no pets""Dammit, I'm mad!

Race carTaco catEvil oliveNever odd or evenUseless knowledge:Aibohphobia- the fear of palindromesRestriction EnzymesRestriction enzymes cleave [cut] both strands of the DNA molecule at certain points called palindromes.

EcoRI recognizes GAATTC segments & cuts them between the G & AcutcutThis produces sticky ends that will readily bond to their sticky compliments.

G A A A C T T G A T C G A A C T T C G G A C G A A C T T G A A C T TForeign DNA cut with the same restriction enzyme G A A C T T G A A C T T G A A A C T T G A T C G A A C T T C G G A CForms recombinant DNASplicingThis is known asRestriction EnzymesBecause the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts.Copies of a DNA molecule will always yield the same set of restriction fragments when exposed to a specific restriction enzyme.

VectorsRecombinant plasmids are produced by splicing restriction fragments from foreign DNA into plasmids.These can be returned relatively easily to bacteria.

VectorsThe original plasmid used to produce recombinant DNA is called a cloning vector, which is a DNA molecule that can carry foreign DNA into a cell and replicate there.

Bacteria cultures also grow quickly, rapidly replicating the foreign genes.Bacteria are most commonly used as host cells for gene cloning because DNA can be easily isolated and reintroduced into their cells.Then, as a bacterium carrying a recombinant plasmid reproduces, the plasmid replicates within it.CloningThe process of cloning a human gene in a bacterial plasmid can be divided into four steps.

Cloning1. Isolation of vector and gene-source DNA. The source DNA comes from human tissue cells.The source of the plasmid is typically E. coli.This plasmid carries an ampR gene, conferring resistance to the antibiotic ampicillin.

Cloning2. Insertion of DNA into the vectorBy digesting both the plasmid and human DNA with the same restriction enzyme we can create thousands of human DNA fragments, one fragment with the gene that we want, and with compatible sticky ends on bacterial plasmids.

Cloning2. Insertion of DNA into the vector

After mixing, the human fragments and cut plasmids form complementary pairs that are then joined by DNA ligase.This creates a mixture of recombinant DNA molecules.

Cloning3. Introduction of the cloning vector into cells.Bacterial cells take up the recombinant plasmids by transformation.This creates a diverse pool of bacteria, some bacteria that have taken up the desired recombinant plasmid DNA, and bacteria that lack the plasmid

Cloning4. Cloning of cells (and foreign genes)We can plate out the transformed bacteria on solid nutrient medium containing ampicillin. Only bacteria that have the ampicillin-resistance plasmid will grow.

Eukaryotic EngineeringMore difficult that prokaryotic engineering due to a nuclear membraneRequires different strategies

Eukaryotic Engineering

Eukaryotic Engineering

Polymersase Chain Reaction (PCR)This technique can quickly amplify any piece of DNA without using cells.

The DNA is incubated in a test tube with special DNA polymerase, a supply of nucleotides, and short pieces of single-stranded DNA as a primer

PCR can make billions of copies of a targeted DNA segment in a few hours.This is faster than cloning via recombinant bacteria.

In PCR, a three-step cycle: heating, cooling, and replication, brings about a chain reaction that produces an exponentially growing population of DNA molecules.

The key to easy PCR automation was the discovery of an unusual DNA polymerase, isolated from bacteria living in hot springs, which can withstand the heat needed to separate the DNA strands at the start of each cycle.

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PCR has amplified DNA from a variety of sources:Fragments of ancient DNA from a 40,000-year-old frozen wooly mammoth.DNA from tiny amount of blood or semen found at the scenes of violent crimes.

PCR has amplified DNA from a variety of sources:DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders

PCR has amplified DNA from a variety of sources:DNA of viral genes from cells infected with difficult-to-detect viruses such as HIV

WARNING!OLD SLIDES AHEADOne indirect method of rapidly analyzing and comparing genomes is Gel Electrophoresis.Comparisons among whole sets of genes and their interactions is the field of Genomics.

Gel electrophoresis separates macromolecules - nucleic acids or proteins - on the basis of their rate of movement through a gel in an electrical field.

*Rate of movement depends on size, electrical charge, and other physical properties of the macromolecules.

For linear DNA molecules, separation depends mainly on size (length of fragment) with longer fragments migrating less along the gel.

Gel Electrophoresis [in a nut shell] EV 1 2 3 4 5Evidencesuspects EV 1 2 3 4 5Who's Guilty?Restriction fragment analysis indirectly detects certain differences in DNA nucleotide sequences.*After treating long DNA molecules with a restriction enzyme, the fragments can be separated by size via gel electrophoresis.

Restriction fragment analysis indirectly detects certain differences in DNA nucleotide sequences.*This produces a series of bands that are characteristic of the nucleotide sequence and that restriction enzyme.

*A normal allele is inserted into somatic cells of a tissue affected by a genetic disorder via a vector [such as a virus].*This alters an afflicted individuals genes.Techniques for gene manipulation hold great potential for treating disease by gene therapy.

Viral DNA is genetically engineered to include the normal gene individuals with Cystic fibrosis lack.

When the virus attacks lung cells, it injects the engineered DNA directly into the lung cells

transforming the lung cells DNA to produce the normal type of mucus.Bone marrow cells, which include the stem cells that give rise to blood and immune system cells, are prime candidates for gene therapy. *A normal allele could be inserted by a viral vector into some bone marrow cells removed from the patient.*If the procedure succeeds, the returned modified cells will multiply throughout the patients life and express the normal gene, providing missing proteins.

By transferring the gene for a protein into a host that is easily grown in culture, one can produce large quantities of normally rare proteins.DNA technology has been used to create many useful pharmaceuticals, mostly proteins.One of the first practical applications of gene splicing was the production of mammalian hormones and other mammalian regulatory proteins in bacteria.* These include human insulin and growth factor.

*Human insulin, produced by bacteria, is superior for the control of diabetes than the older treatment of pig or cattle insulin.*Human growth hormone benefits children with hypopituitarism, a form of dwarfism.

DNA fingerprints can be used forensically to present evidence to juries in murder trials.

This autoradiograph of RFLP bands of samples from a murder victim, the defendant, and the defendants clothes is consistent with the conclusion that the blood on the clothes is from the victim, not the defendant.

The forensics use of DNA fingerprinting extends beyond violent crimes.*For instance, DNA fingerprinting can be used to settle conclusively a question of paternity.

Increasingly, genetic engineering is being applied to environmental work.Scientists are engineering the metabolism of microorganisms to help cope with some environmental problems.*For example genetically engineered microbes that can extract heavy metals from their environments and incorporate the metals into recoverable compounds may become important both in mining materials and cleaning up highly toxic mining wastes.

*In addition to the normal microbes that participate in sewage treatment, new microbes that can degrade other harmful compounds are being engineered.

*Genetically engineered microbes are being used to degrade oil spilled into the oceans.

For many years scientists have been using DNA technology to improve agricultural productivity.*DNA technology is now routinely used to make vaccines and growth hormones for farm animals.To develop a transgenic organism, scientists remove ova from a female and fertilize them in vitro.*The desired gene from another organism are cloned and then inserted into the nuclei of the eggs.*Some cells will integrate the foreign DNA into their genomes and are able to express its protein.To develop a transgenic organism, scientists remove ova from a female and fertilize them in vitro.*The engineered eggs are then surgically implanted in a surrogate mother.*If development is successful, the results is a transgenic animal, containing a genes from a third parent, even from another species.

The pGLO gene that codes for a protein that gives them the ability to glow was inserted into a rabbits ova.

The ova was implanted into a surrogate mother and voila

a glowing bunny was born.

Fluorescent Fish*Because a single transgenic plant cell can be grown in culture to generate an adult plant, plants are easier to engineer than most animals.*These include delayed ripening and resistance to spoilage and disease.Agricultural scientists have engineered a number of crop plants with genes for desirable traits.*These plants may receive genes for resistance to weed-killing herbicides or to infectious microbes and pest insects.*In the past few years, roughly half of the soybeans and corn in America have been grown from genetically modified seeds.Genetic engineering is quickly replacing traditional plant-breeding programs.Round-up Ready Seeds

Scientists are using gene transfer to improve the nutritional value of crop plants.*For example, a transgenic rice plant has been developed that produces yellow grains containing beta-carotene which humans use to produce Vitamin A.

Scientists are using gene transfer to improve the nutritional value of crop plants.*Currently, 70% of children under the age of 5 in Southeast Asia are deficient in vitamin A, leading to vision impairment and increased disease rates.ReviewPolymerase chain reaction (PCR) is a technique used on small quantities of DNA (from a crime scene, for example) to make millions of copies so that the sample can be analysed.DNA profiling is a technique used to identify the origin of a sample of DNA by using gel electrophoresis to match up fragments of the unknown DNA with DNA that has already been identified.The Human Genome Project has succeeded in using DNA sequencers to make a map of all the nitrogenous bases that make up the 46 human chromosomes: this will allow researchers to locate base sequences that might be responsible for genetic diseases, which might then code for beneficial molecules that could be used as medications in the future, or which are shared by different populations thus showing ancestries and migration patterns.

ReviewIn recent decades, scientists have developed laboratory techniques to cut, copy, and paste genes to engineer bacteria, plants, and animals with desirable genetic traits; this is the case with genetically modified Escherichia coli bacteria used to produce human insulin.There are natural and artificial techniques of cloning. Binary fission, budding, and asexual reproduction in plants using techniques such as rooting stem cuttings, result in new individuals that are identical to their parent. Artificial cloning includes reproductive cloning (making a copy of an entire organism) and therapeutic cloning (making copies of certain cells). These laboratory techniques have something in common with genetically engineered organisms: they carry challenging ethical considerations that no previous generation has had to face.

Bacterial Colony Transformation using the pGLO plasmid.It is a fairly simple procedure

Ampicillin sensitive E. coli cells are transferred to cold calcium chloride [CaCl2].

pGLOIt is a fairly simple procedureThe pGLO plasmids [which carry genes for bioluminescence & ampicillin resistance] are added.It is a fairly simple procedureThe bacterial cells are heat shocked at 42C.

This creates small openings in the bacterias plasma membrane.It is a fairly simple procedureSome of the competent cells take up the pGLO plasmid & are transformed.

It is a fairly simple procedureThe cells are cold-shocked to reestablish the bacterias plasma membrane.

It is a fairly simple procedureThe treated cells are spread on agar plates. One plate contains ampicillin.

It is a fairly simple procedureOnly the cells that took up the pGLO plasmid will grow on ampicillin agar.

It is a fairly simple procedureThey will also glow under a black light.

"Transformed"Colonies ofbacteria

All producing the proteins