Download Chapter 20: DNA Technology & Genomics

Chapter 20: DNA Technology
& Genomics
Biotechnology
Manipulation
of organisms/components
to make desired products
Ex. Making wine & cheese with yeast,
selective breeding of organisms,
recombinant DNA products
Genetic
Direct
engineering
gene manipulation
Recombinant DNA (nucleotide sequences
from 2 sources)
Make hormones, & clotting factors
Cloning
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Gene cloning
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Purpose:
Copies of a gene for research
 Produce Protein product
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use bacterial plasmids
Insert desired gene into plasmid
Recombinant plasmid is inserted into
bacterium
Bacterium replicates its genome including the
foreign DNA
Constructing Recombinant DNA
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Restriction enzymes cut DNA at a specific
sequence (4-8 nucleotides) creating
fragments
Cuts leave “sticky ends”
Sticky ends form temporary hydrogen
bonds to complementary sequences
DNA ligase seals bond= stable
recombinant DNA
Identifying Recombinant Genes
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Nucleic Acid Hybridization
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Fluorescent/Radioactively labeled Nucleic acid
probe used
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Probe is complementary to gene of interest
Detection of recombinant gene product
Eukaryotic Cloning
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Yeast cells (eukaryotic fungi) used for
making recombinant genes
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YACs (yeast artificial chromosomes)
Yeast=eukaryotes can post-translationally
modify unlike prokaryotes
Yeast plasmids can carry larger DNA
segments
Yeast must be made competent
electroporation
Polymerase Chain Reaction (PCR)
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Amplifies DNA sample
Procedure
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Denaturation/separation of DNA strands
DNA primers added initiates replication at target
sequence
DNA polymerase adds nucleotides to primers
Cycle is repeated to further amplify DNA sample
DNA Fragment Analysis
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Gel electrophoresis
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Southern blotting
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Gel used as a sieve to separate nucleic acids or
proteins based on size & charge
DNA (-) travels toward + electrode
Long sequences remain toward top; short sequences
move toward bottom banding pattern created
Method which combines gel electrophoresis & nucleic
acid hybridization
Restriction Fragment Length Polymorphisms
(RFLPs)
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Non-coding regions of DNA are analyzed for unique
banding patterns
Identifying Genes & Their Function
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In vitro mutagenesis
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RNA interference (RNAi)
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Mutations introduced inserted back into
cell changes observed
Synthetic double stranded RNA used  blocks
translation
DNA microarray assays
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Genome-wide expression studies
Determines which genes are expressed in a
particular tissue, under which environmental
conditions, & in combination with other genes
Application of DNA Technology
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Medical
Pharmaceutical
Forensic
Environmental
Agricultural
Medical Applications
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Diagnosis of Disease
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PCR & nucleic acid probes used to look for
disease causing mutation even before birth
RFLP markers can be used to detect disease
Gene Therapy
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Alteration of disease affected individual’s
genes
Pharmaceutical Application
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Production of “manufactured” hormones
or other proteins i.e. HGH, insulin, TPA
(dissolves blood clots following heart
attack)
Forensic Evidence
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DNA fingerprint
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RFLP analysis used detects similarities/
differences in DNA
Simple sequence DNA (STRs: simple tandem
repeats) are used to provide markers that are
even more variable between individuals
Environmental Clean-Up Application
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Microorganisms metabolize environmental
waste engineered into other organisms
Used to
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Extract heavy metals
Cleaning up mining waste
Cleaning up oil spills
Cleaning up waste water
Agricultural Application
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Vaccines & growth hormones for farm animals
Transgenic organisms
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Insert cloned gene into the fertilized egg  implant
into surrogate mother
Traits inserted increase nutritional value of animal
products
Genetically engineered plants
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Genes inserted for desirable traits
Ti plasmid most often used vector
Traits= herbicide or pest resistance or increased
nutrients
Debate about GM (genetically modified) organisms