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J .J Genes can be cloned in recombinant plasmids
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J .J Genes can be cloned in recombinant plasmids
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J .J Genes can be cloned in recombinant plasmids
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J . Enzymes are used to ³cut and paste´ DNA
cut DNA at specific
sequences
ƛ Each enzyme binds to DNA at a different
ƛ Many restriction enzymes make staggered cuts that
produce
with single-stranded
ends called Ơsticky endsơ
ƛ Fragments with complementary sticky ends can
associate with each other, forming recombinant DNA
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J .3 Cloned genes can be stored in genomic
libraries
A
is a collection of all of the
cloned DNA fragments from a target genome
¦enomic libraries can be constructed with
different types of vectors
ƛ Plasmid library: genomic DNA is carried by plasmids
ƛ Phage library: genomic DNA is incorporated into
bacteriophage DNA
ƛ Bacterial artificial chromosome (BAC) library:
specialized plasmids can carry large DNA sequences
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J .6 Recombinant cells and organisms can
mass-produce gene products
Cells and organisms containing cloned genes are
used to manufacture large quantities of gene
products
Capabilities of the host cell are matched to the
characteristics of the desired product
ƛ Prokaryotic host: E. coli
ƛ Can produce eukaryotic proteins that do not require post-
translational modification
ƛ Has many advantages in gene transfer, cell growth, and
quantity of protein production
ƛ Can be engineered to secrete proteins
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J .6 Recombinant cells and organisms can
mass-produce gene products
Capabilities of the host cell are matched to the
characteristics of the desired product
ƛ Eukaryotic hosts
ƛ Yeast: S. cerevisiae
ƛ Can produce and secrete complex eukaryotic
proteins
ƛ Mammalian cells in culture
ƛ Can attach sugars to form glycoproteins
ƛ ƠPharmơ animals
ƛ Will secrete gene product in milk
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J .7 CONNECTON: DNA technology has
changed the pharmaceutical industry and
medicine
Products of DNA technology
ƛ Therapeutic hormones
ƛ Insulin to treat diabetes
ƛ Human growth hormone to treat dwarfism
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J .7 CONNECTON: DNA technology has
changed the pharmaceutical industry and
medicine
Products of DNA technology
ƛ *
ƛ Stimulate an immune response by injecting
ƛ Protein from the surface of an infectious agent
ƛ A harmless version of the infectious agent
ƛ A harmless version of the smallpox virus containing
genes from other infectious agents
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J .7 CONNECTON: DNA technology has
changed the pharmaceutical industry and
medicine
Advantages of recombinant DNA products
ƛ Identity to human protein
ƛ Purity
ƛ Quantity
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J .8 CONNECTON: Genetically modified
organisms are transforming agriculture
¦
(¦) organisms contain
one or more genes introduced by artificial means
ë
contain at least one
gene from another species
¦M plants
ƛ Resistance to herbicides
ƛ Resistance to pests
ƛ Improved nutritional profile
¦M animals
ƛ Improved qualities
ƛ Production of proteins or therapeutics
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J .9 Genetically modified organisms raise concerns
about human and environmental health
Scientists use safety measures to guard against
production and release of new pathogens
Concerns related to ¦M organisms
ƛ Can introduce allergens into the food supply
ƛ FDA requires evidence of safety before approval
ƛ Exporters must identify ¦M organisms in food shipments
ƛ May spread genes to closely related organisms
ƛ Hybrids with native plants may be prevented by modifying
¦M plants
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J .J CONNECTON: Gene therapy may
someday help treat a variety of diseases
SCID (severe combined immune deficiency) was
the first disease treated by gene therapy
ƛ First trial in 1990 was inconclusive
ƛ Second trial in 2000 led to the development of
leukemia in some patients due to the site of gene
insertion
Challenges
ƛ Safe delivery to the area of the body affected by the
disease
ƛ Achieving a long-lasting therapeutic effect
ƛ Addressing ethical questions
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DNA PROFILIN¦
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J .JJ The analysis of genetic markers can
produce a DNA profile
is the analysis of DNA fragments
to determine whether they come from a particular
individual
ƛ Compares genetic markers from noncoding regions
that show variation between individuals
ƛ Involves amplification (copying) of markers for
analysis
ƛ Sizes of amplified fragments are compared
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J .J The PCR method is used to amplify DNA
sequences
(
) is a method
of amplifying a specific segment of a DNA
molecule
Relies upon a pair
ƛ Short DNA molecules that bind to sequences at each
end of the sequence to be copied
ƛ Used as a starting point for DNA replication
Repeated cycle of steps for PCR
ƛ Sample is heated to separate DNA strands
ƛ Sample is cooled and primer binds to specific target
sequence
ƛ Target sequence is copied with heat-stable DNA
polymerase
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J .J The PCR method is used to amplify DNA
sequences
Advantages of PCR
ƛ Can amplify DNA from a small sample
ƛ Results are obtained rapidly
ƛ Reaction is highly sensitive, copying only the target
sequence
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J .J3 Gel electrophoresis sorts DNA molecules
by size
¦ separates DNA molecules
based on size
ƛ DNA sample is placed at one end of a porous gel
ƛ Current is applied and DNA molecules move from the
negative electrode toward the positive electrode
ƛ Shorter DNA fragments move through the gel pores
more quickly and travel farther through the gel
ƛ DNA fragments appear as bands, visualized through
staining or detecting radioactivity or fluorescence
ƛ Each band is a collection of DNA molecules of the
same length
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J .J STR analysis is commonly used for DNA
profiling
(
ë
) are genetic
markers used in DNA profiling
ƛ STRs are short DNA sequences that are repeated
many times in a row at the same location
ƛ The number of repeating units can differ between
individuals
ƛ
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compares the lengths of STR
sequences at specific regions of the genome
ƛ Current standard for DNA profiling is to analyze 13
different STR sites
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J .J CONNECTON: DNA profiling has
provided evidence in many forensic
investigations
Forensics
ƛ Evidence to show guilt or innocence
Species identification
ƛ Evidence for sale of products from endangered species
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J .J6 RFLPs can be used to detect differences in
DNA sequences
(
) is a
variation at one base pair within a coding or
noncoding sequence
(
) is a variation in the size of DNA fragments
due to a SNP that alters a restriction site
ƛ RFLP analysis involves comparison of sizes of
restriction fragments by gel electrophoresis
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¦ENOMICS
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J .J7 Genomics is the scientific study of whole
genomes
¦ is the study of an organismƞs complete
set of genes and their interactions
ƛ Initial studies focused on prokaryotic genomes
ƛ Many eukaryotic genomes have since been
investigated
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J .J8 CONNECTON: The Human Genome
Project revealed that most of the human
genome does not consist of genes
Results of the Human ¦enome Project
ƛ Humans have 21,000 genes in 3.2 billion nucleotide
pairs
ƛ Only 1.5 of the DNA codes for proteins, tRNAs, or
rRNAs
ƛ The remaining 88.5 of the DNA contains
ƛ Control regions such as promoters and enhancers
ƛ Unique noncoding DNA
ƛ Repetitive DNA
ƛ Found in centromeres and telomeres
ƛ Found dispersed throughout the genome, related to
that can move or be
copied from one location to another
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J . Proteomics is the scientific study of the full
set of proteins encoded by a genome
ƛ Studies the proteome, the complete set of proteins
specified by a genome
ƛ Investigates protein functions and interactions
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J . J EVOLUTON CONNECTON: Genomes
hold clues to the evolutionary divergence of
humans and chimps
Comparisons of human and chimp genomes
ƛ Differ by 1.2 in single-base substitutions
ƛ Differ by 2.7 in insertions and deletions of larger
DNA sequences
ƛ Human genome shows greater incidence of
duplications
ƛ ¦enes showing rapid evolution in humans
ƛ ¦enes for defense against malaria and tuberculosis
ƛ ¦ene regulating brain size
ƛ FOXP2 gene involved with speech and vocalization
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