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BiotechnologyPacket #12
Chapter #9
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Introduction
Since the 1970’s, humans have been attempted to manipulate and modify genes in a way that was somewhat predictable.
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Introduction II
Scientists would select a gene to be inserted into an organism
Cut two DNA molecules into fragments using restriction enzymes
Splice the fragments together into the desired combination Producing recombinant DNA
Introduce the new DNA into a living cell for replication
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Introduction III
Recombinant DNA technology isolates and amplifies specific sequences of DNA by incorporating them into vector DNA molecules.
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The Players Involved in the Making of Recombinant DNA
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Introduction
Recombinant DNA is made by splicing a DNA fragment of interest into a small quickly dividing replicating molecule (plasmid).
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Donor & Transgenic Organisms
The organism providing the DNA is called the donor.
After recombination, an organism that contains an artificially inserted, foreign piece of DNA, is called a transgenic organism.
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The Importance of Transgenic Organisms
Transgenic organisms allow gene targeting and mutagenesis screening that help identify the function of a gene and its protein product.
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Vectors
Vectors, normally in the form of plasmids, is a genome into which the DNA fragments, removed from the donor, are inserted.
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Restriction Enzymes
Enzymes that are used to cut DNA into specific fragments.
Each restriction enzyme recognizes and cuts DNA at a highly specific base sequence.
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The Making of Transgenic Organisms
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The Making of a Transgenic Organism
The DNA of interest is excised, from the donor, using “scissors” known as a restriction enzyme. The excised DNA is
called a DNA fragment.
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The Making of a Transgenic Organism
The DNA fragment is inserted into the vector via one of multiple methods.
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The Making of a Transgenic Organism
Once inserted, DNA ligase is used to join the DNA fragment together with the vector’s genome.
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The Making of a Transgenic Organism
The new transgenic organism is duplicated.
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Vectors Currently Under Study
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Vectors Under Study…
Vectors currently under study includeRetrovirusesAdenovirusesHerpes simplex virusRhinovirusHuman Immunodeficiency Virus (HIV)
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Genomics & Genetic Libraries
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Genomic Library & cDNA Library
Genomic Library DNA library containing an
organism’s complete genome In the form of thousands
of DNA fragments
cDNA Library DNA library made up of
“DNA clones” reconstructed using reverse transcriptase Must be made from mRNA
Genomics Sub-discipline in genetics of
characterizing the entire genomes of organisms.
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Homework Assignment
What are some of the advantages, and disadvantages, of having a cDNA library?
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Genetic Probes
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Genetic Probes
Genetic probes are radioactively labeled DNA or RNA sequence that enables geneticists to identify complementary nucleic acid sequences. If used to identify a DNA
strand, the DNA molecule will have to be separated into into two strands via artificial denaturation—heat.
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The Making of Genetic ProbesSouthern Blot Technique
DNA fragments, produced using restriction enzymes, are separated via gel electrophoresis. Fragments are blotted
onto a nitrocellulose or nylon membrane.
The membrane is bathed in a labeled probe for a specific DNA fragment.
The selected DNA fragments are cut out of the gel
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Homework Assignment
Define Northern Blot.
Define Western Blot.
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Making Copies of DNA in a Lab Setting
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Introduction
Once a sequence of DNA (DNA fragment) has been isolated, it is sometimes necessary to make large amounts of that sequence for study.
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Polymerase Chain Reaction
Allows rapid, efficient amplification of DNA sequences of interest. In vitro technique
Researchers target a particular DNA sequence, by specific primers, and then clone the DNA sequence by heat resistant DNA polymerase.
Used to help amplify DNA from crime scenes and archaeological remains
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Gene Therapy
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Gene Therapy
Simple idea—hard to practice
The use of sequencing, cloning and vector insertion techniques to deliver working versions of genes to individuals who are born with deleterious mutant versions of the gene. Germ Line Therapy Somatic Gene Therapy
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Genetic Engineering & Food
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Genetic Engineering of Agricultural Species
Foreign genes, under study, for insertion into commercial plant species. Helps provide Selective herbicide resistance Increased yield Plant-grown vaccines and
pharmaceuticals Improved nutrient balance
Problems? Human allergic reactions to
foreign proteins Increased use of herbicides “jumping” of plasmids from
commercial crops to weed species.
Eco-mayhem!
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Review