Chapter 20:
DNA Technology
 Genetic Engineering
 Manipulation of DNA
 Recombinant DNA
 DNA from two different sources
 Biotechnology
 Manipulation of organisms to make
useful products
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http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapte
r14/animation_quiz_6.html
How can plasmids help us?
 A way to get genes into bacteria easily



insert new gene into plasmid (recombinant DNA)
insert plasmid into bacteria = vector
bacteria now expresses new gene
 Production of many copies of inserted gene
transformed
 bacteria make “new” protein
gene from
other organism
cut DNA
plasmid
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recombinant
plasmid
+
vector
glue DNA
bacteria
Grow bacteria…make clones
gene from
other organism
recombinant
plasmid
+
vector
plasmid
grow
bacteria
harvest (purify)
protein
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transformed
bacteria
Bacterium
Cell containing gene
of interest
1 Gene inserted
into plasmid
Overview of
gene cloning
with a bacterial
plasmid,
showing
various uses of
cloned genes
Figure 20.2
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Plasmid
Bacterial
chromosome
Gene of
interest
DNA of
chromosome
Recombinant
DNA (plasmid)
Recombinate
bacterium
2 Plasmid put into
bacterial cell
Gene of
interest
Protein expressed
by gene of interest
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
3 Host cell grown in culture,
3 form a clone of cells
to
containing the “cloned”
gene of interest
Protein harvested
Basic
research
on protein
4 Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Human growth
hormone treats
stunted growth
cut DNA at specific sequences CTGAATTCCG
 restriction site
GACTTAAGGC



Restriction enzymes
 Action of enzyme
 produces restriction fragments

produces protruding ends
 sticky ends
CTG|AATTCCG
GACTTAA|GGC
 will bind to any complementary
DNA

DNA ligase is an enzyme
 Seals the bonds between restriction fragments
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Restriction enzymes
 Cut DNA at specific sites

leave “sticky ends”
restriction enzyme cut site
GTAACGAATTCACGCTT
CATTGCTTAAGTGCGAA
restriction enzyme cut site
GTAACG AATTCACGCTT
CATTGCTTAA GTGCGAA
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Sticky ends
 Cut other DNA with same enzymes


leave “sticky ends” on both
can glue DNA together at “sticky ends”
GTAACG AATTCACGCTT
CATTGCTTAA GTGCGAA
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gene
you want
GGACCTG AATTCCGGATA
CCTGGACTTAA GGCCTAT
chromosome
want to add
gene to
GGACCTG AATTCACGCTT
CCTGGACTTAA GTGCGAA
combined
DNA
Sticky ends help glue genes together
cut sites
gene you want
cut sites
TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTT
AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA
sticky ends
AATTCTACGAATGGTTACATCGCCG
GATGCTTACCAATGTAGCGGCTTAA
isolated gene
cut sites AATTCTACGATCGCCGATTCAACGCTT
chromosome want to add gene to
AATGGTTACTTGTAACG
TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA
DNA ligase joins the strands
sticky ends stick together
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Recombinant DNA molecule
chromosome with new gene added
TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC
CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC
Many uses of restriction enzymes…
 Now that we can cut DNA with
restriction enzymes…
we can cut up DNA from different
people… or different organisms…
and compare it
 why?

 forensics
 medical diagnostics
 paternity
 evolutionary relationships
 and more…
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Uses of genetic engineering
 Genetically modified organisms (GMO)

enabling plants to produce new proteins
 Protect crops from insects: BT corn
 corn produces a bacterial toxin that kills corn
borer (caterpillar pest of corn)
 Extend growing season: fishberries
 strawberries with an anti-freezing gene from
flounder
 Improve quality of food: golden rice
 rice producing vitamin A
improves nutritional value
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Jelly fish “GFP”
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Transformed vertebrates
Copy DNA without plasmids? PCR!
 Polymerase Chain
Reaction

method for making
many, many copies
of a specific
segment of DNA
 What do you need?



template strand
DNA polymerase
enzyme
nucleotides
 ATP, GTP, CTP, TTP
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primer
PCR process
 What do you need to do?



in tube: DNA, DNA polymerase enzyme, primer, nucleotides
denature DNA: heat (90°C) DNA to separate strands
anneal DNA: cool to hybridize with primers (H-bonds) & build DNA 5’3’ (extension)
What does 90°C
do to our
DNA polymerase?
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play DNAi movie
The polymerase problem
 Heat DNA to denature (unwind) it
90°C destroys DNA polymerase
 have to add new enzyme every cycle

 almost impractical!
 Need enzyme that can
withstand 90°C…

Taq polymerase
 from hot springs bacteria
 Thermus aquaticus
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Comparing cut up DNA
 How do we compare DNA fragments?

separate fragments by size
 How do we separate DNA fragments?

run it through a gelatin
 agarose
 made from algae

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gel electrophoresis
Gel electrophoresis
 A method of separating DNA
in a gelatin-like material
using an electrical field
DNA is negatively charged
 when it’s in an electrical
field it moves toward the
positive side

–
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DNA 

+
“swimming through Jello”
Gel electrophoresis
 DNA moves in an electrical field…

so how does that help you compare DNA
fragments?
 size of DNA fragment affects how far it travels
 small pieces travel farther
 large pieces travel slower & lag behind
–
DNA 

+
“swimming through Jello”
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fragments of DNA
separate out based
on size
Running a gel
cut DNA with restriction enzymes
1
2
Stain DNA


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ethidium bromide
binds to DNA
fluoresces under
UV light
3
Uses: Evolutionary relationships
 Comparing DNA samples from different
organisms to measure evolutionary
relationships
turtle snake rat squirrel fruitfly
–
DNA

+
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1
2
3
4
5
1
2
3
4
5
Uses: Medical diagnostic
 Comparing normal allele to disease allele
chromosome
with normal
allele 1
chromosome with
disease-causing
allele 2
–
DNA

Example: test for Huntington’s disease
+
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Uses: Forensics
 Comparing DNA sample from crime
scene with suspects & victim
suspects
S1 S2 S3
crime
scene
V sample
–
DNA

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+
Differences at the DNA level
 Why is each person’s DNA pattern different?

sections of “junk” DNA
 doesn’t code for proteins
 made up of repeated patterns
 CAT, GCC, and others
 each person may have different number of repeats
 many sites on our 23 chromosomes with
different repeat patterns
GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTT
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CGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA
Human Genome Project
On June 26, 2001, HGP published the “working
draft” of the DNA sequence of the human genome.
Historic Event!
blueprint
of a human
 the potential to
change science &
medicine

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TACGCACATTTACGTACGCGGATGCCGCGACTATGATC
ACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACT
human genome (2003)
CGACTAGCATGATCGATCAGCTACATGCTAGCACACYC
GTACATCGATCCTGACATCGACCTGCTCGTACATGCTA
3.2
billion
bases
CTAGCTACTGACTCATGATCCAGATCACTGAAACCCTA
GATCGGGTACCTATTACAGTACGATCATCCGATCAGAT
CATGCTAGTACATCGATCGATACTGCTACTGATCTAGC
TCAATCAAACTCTTTTTGCATCATGATACTAGACTAGC
TGACTGATCATGACTCTGATCCCGTAGATCGGGTACCT
ATTACAGTACGATCATCCGATCAGATCATGCTAGTACA
TCGATCGATACTGCTACTGATCTAGCTCAATCAAACTC
TTTTTGCATCATGATACTAGACTAGCTGACTGATCATG
ACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGA
TCATCCGATCAGATCATGCTAGTACATCGATCGATACT
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Advancements to sequencing
 Fluorescent tagging sequence data
 Computer read & analyzed
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 An alternative approach to sequencing
whole genomes starts with the sequencing
of random DNA fragments

Powerful computer programs would then
assemble the resulting very large number of
overlapping short sequences into a single
continuous sequence
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