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Chapter 20
DNA Technology & Genomics
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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Page 2
 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
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