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Chapter 20 – DNA
Technology and
Genomics
Recombinant DNA
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Def: DNA in which
genes/nucleotide
sequences from 2
different sources are
combined in vitro into the
same DNA molecule
Genetic engineering:
direct manipulation of
genes for practical
purposes
Biotechnology:
manipulation of organisms
or their components to
perform practical tasks or
provide useful products
DNA Cloning
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Permits the
production of
multiple copies
of a specific
gene or DNA
segment
Most practical
applications
utilize bacteria
and bacterial
plasmids
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Overview
of gene
cloning
with a
bacterial
plasmid
Creating Recombinant DNA
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Restriction enzymes
(endonucleases): in nature,
these enzymes protect bacteria
from intruding DNA; they cut up
the DNA (restriction); very
specific (flash movie)
Restriction site: recognition
sequence for a particular
restriction enzyme
Restriction fragments: segments
of DNA cut by restriction
enzymes in a reproducible way
Sticky end: short extensions of
restriction fragments
DNA ligase: enzyme that can
join the sticky ends of DNA
fragments
Cloning vector: DNA molecule
that can carry foreign DNA into a
cell and replicate there (usually
bacterial plasmids
Restriction Enzymes
Cloning
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Cloning of
human DNA
using a
bacterial
plasmid
Same
technique
used to create
plasmid for our
transformation
lab
http://www.dna
i.org/b/index.ht
ml
Cloning a Gene
Identifying Clone Cells
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How identify cells?
Insert ampicillin resistance gene (ampr) into plasmid, culture cells
on ampicillin, if live, have incorporated plasmid
Nucleic acid hybridization, uses radioactive nucleic acid probe
Genomic Libraries
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Can store these genes in bacteria or viruses to
create a genomic library
a cDNA or complimentary DNA library is made
in vitro by reverse transcription of all the mRNA
produced by a particular cell
cDNA is used to create plasmids
Rainbow (donor) and CC (clone)
Organismal Cloning
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Producing one or more organisms that
are genetically identical to the parent
that donated the single cell
Important because can generate stem
cells – unspecialized cells that can
reproduce indefinitely and differentiate
into specialized cells
Stem cells have great potential for
regenerating damaged tissues
Cloning
Technique
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Technique
used to
create/clone
Dolly, the first
mammal ever
clonned.
Stem Cells
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Difference
between
adult and
embryonic
stem cells
DNA Anaylsis and Genomics
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Gel Electrophoresis
Restriction Fragment Length
Polymorphism or RFLP analysis
Southern Blot
Polymerase Chain Reaction or PCR
DNA Sequencing
DNA microarray of gene expression
Gel Electrophoresis
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Used to separate nucleic acids or
proteins that differ in size or
electrical charge
Nucleic acids carry a negative
charge (why?) so they will move
towards the positive end of the
porous gel
1. samples loaded in gel at negative
end
2. an electrical current “pulls” the
fragments towards the positive end,
smaller fragments move further
than larger ones
DNA binding dye used to visualize
the fragments
Restriction Fragment Length
Analysis
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Useful in comparing
two different DNA
molecules, like 2
alleles of a gene
If have a small
change in bp
sequence, restriction
enzymes will cut at
different places
producing different
fragments
Example: β-globin
allele (sickle-cell)
Restriction Fragment Length
Analysis (cont.)
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What if when comparing different genomic
DNA there are too many bands for analysis?
Use southern blotting
Southern Blot – combines gel electrophoresis
and nucleic acid hybridization (next slide)
RFLP (restriction length polymorphism) –
differences in the restriction sites on
homologous chromosomes that result in
different restriction fragment patterns, found
in noncoding regions of the DNA; because
these are inherited they can be used as
genetic markers for making linkage maps
RFLP and Southern Blotting
PCR
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Polymerase Chain
Reaction
Method of amplifying
samples of DNA in
vitro
Requires: dsDNA,
heat-resistant DNA
polymerase, RNA
primers, DNA
nuclotides
After 20+ cycles
target DNA vast
outnumbers all other
DNA sequences
http://www.dnai.org/
b/index.html
Genome Mapping
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Human Genome Project
– completed in 2003
3 step approach
– Genetic (linkage) mapping
– uses recombination
frequencies, the ordering
of genetic markers like
RFLPs
– Physical mapping – cutting
the DNA with restriction
enzymes and putting them
in order, make fragments
overlap and use probes
– DNA sequencing
DNA
Sequencing
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Dideoxy chaintermination
method
ssDNA incubated
with tagged
nucleotides
DNA synthesis is
stopped with
tagged nucleotide
Labeled strands
separated through
a gel and analyzed
with a
fluorescence
detector
http://www.dnai.or
g/b/index.html
Gene
Expression
Levels
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DNA microassay
– can test
thousands of
genes
simultaneously to
determine which
are expressed at
any one time
(tissue type,
environmental
conditions,
stages of
development,
etc…)
DNA micro assay cont.
Applications of DNA Technology
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Medical
– Diagnosis of diseases
– Human gene therapy
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Pharmaceutical Products
Forensic Evidence
Environmental Cleanup
Agricultural
– Animal husbandry
– Genetic engineering in plants