Chapter 20
Biotechnology
Bacteria
 Bacteria review
one-celled prokaryotes
 reproduce by mitosis

 binary fission

rapid growth
 generation every ~20 minutes
 108 (100 million) colony overnight!
dominant form of life on Earth
 incredibly diverse

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Bacterial genome
 Single circular chromosome
haploid
 naked DNA

 no histone proteins

~4 million base pairs
 ~4300 genes
 1/1000 DNA in eukaryote
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Transformation
 Bacteria are opportunists

pick up naked foreign DNA
wherever it may be hanging out
 have surface transport proteins that are
specialized for the uptake of naked DNA


mix heat-killed
pathogenic &
non-pathogenic
bacteria
import bits of chromosomes from
other bacteria
incorporate the DNA bits into their
own chromosome
 express new genes
 transformation
 form of recombination
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mice die
Plasmids
 Small supplemental circles of DNA
 5000 - 20,000 base pairs
 self-replicating

carry extra genes
 2-30 genes
 genes for antibiotic resistance

can be exchanged between bacteria
 bacterial sex!!
 rapid evolution

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can be imported from
environment
How can plasmids help us?
 A way to get genes into bacteria easily
insert new gene into plasmid
 insert plasmid into bacteria = vector
 bacteria now expresses new gene

 bacteria make new protein
gene from
other organism
cut DNA
plasmid
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recombinant
plasmid
+
vector
glue DNA
transformed
bacteria
Biotechnology
 Plasmids used to insert new genes into bacteria
cut DNA
gene we
want
like what?
…insulin
…HGH
…lactase
cut plasmid DNA
ligase
recombinant
APplasmid
Biology
insert “gene we want”
into plasmid...
“glue” together
How do we cut DNA?
 Restriction enzymes
restriction endonucleases
 discovered in 1960s
 evolved in bacteria to cut up foreign DNA

 “restrict” the action of the attacking organism
 protection against viruses
& other bacteria
 bacteria protect their own DNA by methylation &
by not using the base
sequences recognized
by the enzymes
in their own DNA
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What do you notice about these phrases?
radar
palindromes
racecar
Madam I’m Adam
Able was I ere I saw Elba
a man, a plan, a canal, Panama
Was it a bar or a bat I saw?
go hang a salami I’m a lasagna hog
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cut DNA at specific sequences CTGAATTCCG
 restriction site
symmetrical “palindrome”
 produces protruding ends
GACTTAAGGC



Restriction enzymes
 Action of enzyme
Madam I’m Adam

 sticky ends
CTG|AATTCCG
GACTTAA|GGC
 will bind to any complementary DNA
 Many different enzymes

named after organism they are found in
 EcoRI, HindIII, BamHI, SmaI
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1960s | 1978
Discovery of restriction enzymes
Werner Arber
Daniel Nathans
Restriction enzymes are
named for the organism
they come from:
EcoRI = 1st restriction
enzyme found in E. coli
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Hamilton O. Smith
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
AATTCTACGAATGGTTACATCGCCG
GATGCTTACCAATGTAGCGGCTTAA
sticky ends
cut sites
isolated gene
chromosome want to add gene to
AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTT
TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA
DNA ligase joins the strands
sticky ends stick together
Recombinant DNA molecule
chromosome with new gene added
TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC
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CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC
Why mix genes together?
 Gene produces protein in different
organism or different individual
human insulin gene in bacteria
TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC
CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC
“new” protein from organism
ex: human insulin from bacteria
aa aa aa aa aa aa aa aa aa aa
bacteria
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human insulin
The code is universal
 Since all living
organisms…



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use the same DNA
use the same code
book
read their genes
the same way
Copy (& Read) DNA
 Transformation
insert recombinant plasmid
into bacteria
 grow recombinant bacteria in agar cultures

 bacteria make lots of copies of plasmid
 “cloning” the plasmid
production of many copies of inserted gene
 production of “new” protein

 transformed phenotype
DNA  RNA  protein  trait
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Grow bacteria…make more
gene from
other organism
recombinant
plasmid
+
vector
plasmid
grow
bacteria
harvest (purify)
protein
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transformed
bacteria
Fig. 20-5a
Foreign genome
cut up with
restriction
enzyme
or
Recombinant
phage DNA
Bacterial
clones
(a) Plasmid library
Recombinant
plasmids
(b) Phage library
Phage
clones
 A library is made by cloning DNA made in

vitro by reverse transcription of all the
mRNA produced by a particular cell
A cDNA library represents only part of the
genome—only the subset of genes
transcribed into mRNA in the original cells
Fig. 20-6-1
DNA in
nucleus
mRNAs in
cytoplasm
Complementary
DNA (cDNA)
Fig. 20-6-2
DNA in
nucleus
mRNAs in
cytoplasm
Complementary
DNA (cDNA)
mRNA
Reverse
transcriptase Poly-A tail
DNA Primer
strand
Fig. 20-6-3
DNA in
nucleus
mRNAs in
cytoplasm
Complementary
DNA (cDNA)
mRNA
Reverse
transcriptase Poly-A tail
Degraded
mRNA
DNA Primer
strand
Fig. 20-6-4
DNA in
nucleus
mRNAs in
cytoplasm
Complementary
DNA (cDNA)
mRNA
Reverse
transcriptase Poly-A tail
Degraded
mRNA
DNA
polymerase
DNA Primer
strand
Fig. 20-6-5
DNA in
nucleus
mRNAs in
cytoplasm
Complementary
DNA (cDNA)
mRNA
Reverse
transcriptase Poly-A tail
DNA Primer
strand
Degraded
mRNA
DNA
polymerase
cDNA
Fig. 20-8a
Amplifying DNA in Vitro: The Polymerase
Chain Reaction (PCR)
5
TECHNIQUE
3
Target
sequence
Genomic DNA
3
5
Fig. 20-8b
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Fig. 20-8c
Cycle 2
yields
4
molecules
Fig. 20-8d
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Fig. 20-8
5
TECHNIQUE
3
Target
sequence
3
Genomic DNA
1 Denaturation
2
5
5
3
3
5
Annealing
Cycle 1
yields
2
molecules
Primers
3
Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Fig. 20-9a
TECHNIQUE
Mixture of
DNA molecules of
different
sizes
Southern Blotting
Power
source
– Cathode
Anode +
Gel
1
Power
source
–
+
Longer
molecules
2
Shorter
molecules
Fig. 20-9b
RESULTS
Southern Blotting
Fig. 20-10
Southern Blotting
Normal -globin allele
175 bp
DdeI
Sickle-cell
allele
Large fragment
201 bp
DdeI
Normal
allele
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
376 bp
DdeI
201 bp
175 bp
Large fragment
376 bp
DdeI
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of -globin gene
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
Fig. 20-11a
Southern Blotting
TECHNIQUE
DNA + restriction enzyme
Restriction
fragments
I
II III
Nitrocellulose
membrane (blot)
Heavy
weight
Gel
Sponge
I Normal II Sickle-cell
allele
-globin
allele
III Heterozygote
1 Preparation of restriction fragments
Alkaline
solution
2 Gel electrophoresis
Paper
towels
3 DNA transfer (blotting)
Fig. 20-11b
Southern Blotting
Radioactively labeled
probe for -globin gene
I
II III
Probe base-pairs
with fragments
Fragment from
sickle-cell
-globin allele
Fragment from
normal -globin
Nitrocellulose blot
allele
4 Hybridization with radioactive probe
I
II III
Film
over
blot
5 Probe detection
DNA Sequencing
Fig. 20-12a
TECHNIQUE
DNA
(template strand)
Primer
Deoxyribonucleotides
Dideoxyribonucleotides
(fluorescently tagged)
dATP
dCTP
ddATP
ddCTP
dTTP
DNA
polymerase
dGTP
ddTTP
ddGTP
DNA Sequencing
Fig. 20-12b
TECHNIQUE
DNA (template
strand)
Labeled strands
Shortest
Direction
of movement
of strands
Longest
Longest labeled strand
Detector
Laser
RESULTS
Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
Shortest labeled strand
Fig. 20-13
TECHNIQUE
1 cDNA synthesis
mRNAs
cDNAs
2 PCR amplification
Primers
-globin
gene
3 Gel electrophoresis
RESULTS
Embryonic stages
1 2 3 4 5
6
Reverse
transcriptase
polymerase
chain reaction
(RT-PCR)
You should now be able to:
1. Describe the natural function of restriction
2.
3.
4.
enzymes and explain how they are used in
recombinant DNA technology
Outline the procedures for cloning a
eukaryotic gene in a bacterial plasmid
Define and distinguish between genomic
libraries using plasmids, phages, and
cDNA
Describe the polymerase chain reaction
(PCR) and explain the advantages and
limitations of this procedure
5. Explain how gel electrophoresis is used to
6.
7.
8.
analyze nucleic acids and to distinguish
between two alleles of a gene
Describe and distinguish between the
Southern blotting procedure, Northern
blotting procedure, and RT-PCR
Distinguish between gene cloning, cell
cloning, and organismal cloning
Describe how nuclear transplantation was
used to produce Dolly, the first cloned
sheep
9. Describe the application of DNA
technology to the diagnosis of genetic
disease, the development of gene therapy,
vaccine production, and the development
of pharmaceutical products
10.Define a SNP and explain how it may
produce a RFLP
11.Explain how DNA technology is used in
the forensic sciences