Chapter 20
DNA Technology & Genomics
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Biotechnology Terms
Biotechnology Process of manipulating organisms or their components to
make useful products Genetic engineering + tissue/cell culturing technologies
Genetic Engineering Manipulation of individual genes or entire genomes Insulin (insulin E. coli bacteria OR yeast) & GMO
(Genetically Modified Organism)
Recombinant DNA Artificially created DNA Typically, DNA is integrated from another species
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Biotechnology Terms (Page 2)
Gene Cloning Laboratory production of multiple copies of DNA segment Therapeutic cloning – embryonic stem cells
Spinal cord injuries Reproductive (organismal) cloning – Dolly the sheep
Restriction Enzymes Enzymes that cut DNA at specific locations Usually, derived from bacteria Cut sites of DNA = restriction fragments Sticky ends – restriction fragments usually have one end
longer than the other
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Quick Assignment
Relate the 6 terms just discussed in a concept map.
Be prepared to defend your arrangement
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Cloning Process
5 steps (first 2)1. Identify & isolate the gene of interest
Involves finding a cloning vector – plasmid or organism used to carry the DNA sequence to be cloned
2. Cut gene of interest from original site & open up vector’s DNA using a ________ ________ This ensures matching sticky ends on gene of interest &
vector DNA
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Cloning Process (Page 2)
5 steps (3-4)3. Combine the 2 DNA pieces (into a recombinant plasmid?)
Recombinant plasmid – plasmid + DNA fragments Sealed together using DNA Ligase Remember: we used ________ ________ to cut gene of
interest from original site & cut vector’s DNA This ensures matching sticky ends on gene of interest &
vector DNA
4. Transfer the vector (recombinant plasmid) into a host cell Usually involves bacterial transformation
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Bacteria & Genetic Recombination
Conjugation Bacterial Sex Genetic material is exchanged
by direct contact
Transduction Phage transfer of DNA Involves a phage vector
Phage moves the DNA from bacterium to other bacterium
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Bacteria & Genetic Recombination
Transformation Uptake of exogenous
DNA Griffith’s experiment -
pathogenic DNA was transferred to benign bacteria
Most common method for genetic engineering
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Step 5
Select for transformed cells Link the gene of interest with
a reporter gene Such as pBLU or pGLO pBLU = Blue coloration pGLO = fluorescent green
under UV light
In Lab 6, we will insert the coloration gene and an ampicillin resistance gene to select for transformed cells
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At this point…
You know which cells have the gene of interest
You can identify the cells that have the gene of interest
Now what?
You need to extract the gene of interest
How would you do that?
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Nucleic Acid Hybridization
Detects the gene of interest
Uses a short, single stranded DNA or RNA called a nucleic acid probe
The nucleic acid probe is complementary to a known sequence in the gene of interest
Usually attach a radioactive isotope or fluorescent tag protein so that it is detectable
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Genomic Libraries
Nucleic Acid Hybridization repeated many times produces a genomic library Thousands of recombinant clones Each has a piece of the original genome being studied
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cDNA Library
cDNA = complementary DNA
mRNA is extracted from cells
Use what enzyme to make DNA from this mRNA?
Then make another strand of DNA using what enzyme?
cDNA library is only a portion of the genome Portion that codes for mRNA Exons? Introns? tRNA? rRNA?
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Microarray Assay
Genome-wide study of gene expression
Different genes are in each well
Identifies gene interactions + provides clues to gene functions
Take samples throughout development + assay to determine which genes are expressed and at what stages Detect patterns of expression throughout development Detect likely response to a pathogenic agent
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PCR Polymerase Chain Reaction Thermal cycling Amplification of DNA
3 Steps Denaturation (Heating)
Annealing (Cooling)Primer formation
Extension DNA polymerase adds nucleotides at 3’ end
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Gel Electrophoresis DNA is negatively charged so it moves AWAY from the (-) cathode toward the (+) anode
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Southern Blotting
Used to detect specific DNA sequences
Useful for comparing samples
Combines gel electrophoresis + nucleic acid hybridization
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DNA Technology affects us…
Disease Diagnosis PCR used to detect traces of viral DNA or RNA in sample RFLP (Restriction Fragment Length Polymorphisms)
Different alleles have different RFLPs
Gene Therapy – alter afflicted genes
Pharmaceutical Production – Insulin production
Forensic Application – DNA fingerprints
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Environmental cleanup Genetically engineered microbes Detoxification of specific wastes
Agricultural applications Insert pest-resistant or drought-resistant genes GMO (Genetically Modified Organisms)
You eat GMO corn, soybeans, canola and cottonseed oil Probably at least weekly 46% of GMOs are grown in US Europe had 12 year moratorium on growing GE foods