Chapter 9 Study Guide
Define and Recognize:
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Clone: Population of cells arising from 1 cell; Each carry a new gene
PCR: Polymerase Chain Reaction; Rapid process that makes billions of copies of a gene;
Makes multiple copies of piece of DNA enzymatically
Bt Toxin: Bacillus Thuringiensis toxin; Biological insecticide; Bt toxin is produced by B.
Thuringiensis and is inserted into some plants and when insects eat it they die.
Reverse Transcriptase: an enzyme used to generate complementary DNA (cDNA) from
an RNA template
Biotechnology and Recombinant DNA
Biotechnology: use of microorganism cells or cell components to make a products; Examples
include food, antibiotics, vitamins and enzymes
Recombinant DNA technology (rDNA): insertion or modification of genes to produce desired
proteins
Vector: self-replicating DNA used to carry desired gene to new cell; Example of a vector is a
plasmid;
Clone: population of cell arising from one cell; each carries a new gene
Figure 9.1A Modification Procedure (Study Slide)
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Take vector (like a plasmid) and recombine with DNA that has desired gene
Put vector (plasmid) into bacteria
Cells with genes of interest are cloned
Purpose: To create and harvest copies of genes; create and harvest protein production
of gene
Restriction Enzymes: Study Figure 9.2
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To get into vector, they cut sequences of DNA
Destroy bacteriophage DNA in bacterial cells
Cannot destroy (host) Dna with methylated cytosine’s
Figure 9.3: Plasmid (vector) used for cloning
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Plasmid normally has 3 important things:
o Reaction enzyme sites
o ORI: origin of replication
o ampR: Antibiotic resistance gene
Polymerase Chain Reaction (PCR) = routine method of DNA amplification
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Rapid process to make billions of copies of a gene; make multiple copies of a piece of a
DNA enzymatically; technique where small samples of DNA can be quickly amplified
Used to:
o clone DNA for recombination
o amplifies DNA to detectable levels
o sequence DNA
o Diagnose genetic disease
o Detect pathogens
Figure 9.4 PCR: 3 steps to the polymerase chain reaction
o Melt Step: Melt DNA at 94C; This breaks Dna apart and becomes single strand
DNA
o Amealing Step: At 60C, primers attach to single strand DNA
o Extension: At 72C, We have synthesized new DNA strand with Dna polymerase
that’s heat stable, called O TAQ DNA Polymerase; Builds new DNA molecule
o This process takes 1 dna strand and makes 2, 4, 6, 8, etc.
Figure 9.8 Genomic libraries (less used method to obtain DNA)
o It takes DNA from organism of interest
o Digests with reaction enzymes
o Makes thousands of clones each with different DNA fragments = “libraries”
Obtaining DNA
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Complementary DNA (cDNA): is made from mRNA by reverse transcriptase
Figure 9.9 Making cDNA for Eukaryotic Gene
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Isolate mRNA
Make cDNA copy using reverse transcriptase ( which comes from retroviruses)
No introns= got rid of them
Figure 9.11 Blue-white Screening (One method of selecting recombinant bacteria)
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Selection: Use of antibiotic to inhibit bacteria that don’t carry ampicillin resistance gene
on vector
Screening: Restriction enzyme site is in LAC-Z gene; insertion of DNA of interest mutates
LAC-Z gene
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Only bacteria that picked up the plasmid will grow in the presence of ampicillin;
X-Gal: Dye that becomes blue in the presence of LAC-Z
So take the vector and DNA- insert in E.Coli- add to agar with ampicillin and x-gal; you
will get some blue colonies and some white colonies
o Blue Colonies: Make LAC-Z; Don’t make DNA of interest
o White Colonies: No LAC-Z; Do have DNA of interest
Making a Product (If we got DNA into vector, and all checks out)
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E. Coli (used to synthesizes gene products):
o Easily grown and known genomics
o Need to eliminate endotoxin from products
o Cells need to be lysed
Saccharomyces cerevisiae (yeast) :
o Easily grown and known genomics
o May express eukaryotic genes easily
Plant cells and whole plants:
o May express eukaryotic genes easily
o Plants are easily grown
Mammalian cells:
o May express eukaryotic cells easily
o Harder to grow
Therapeutic Applications (How we use it)
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Human enzymes and other proteins
Subunit vaccines
Nonpathogenic viruses carrying genes for pathogen’s antigens as DNA vaccines
Gene Therapy to replace defective or missing genes
Example of application is Hep B Vaccine
Scientific Applications
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Understanding DNA
Sequencing organisms’ genomes
DNA fingerprinting for identification (track infectious diseases and where the outbreak
originated)
Agrobacterium: Engineering bacteria for agriculture; well known for its ability to transfer DNA
between itself and plants;
Some benefits include:
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Bt toxin
Herbicide resistance
Suppression of genes: Antisense DNA
Nutrition
Human Proteins