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Chapter 13

Biotechnology
Chapter2 13
Traditional Applications

Biotechnology is applied biology


• Modern focus on genetic
engineering, recombinant DNA
technology, and analysis of
biomolecules
Chapter3 13
Traditional Applications

Traditional (historical)
applications of biotechnology
date back to over 10,000
years ago
• Use of yeast to produce beer
and wine in Egypt and Near East
• Selective breeding of plants
• Selective breeding of animals
Chapter4 13
Genetic Engineering

Genetic engineering refers to the


modification of genetic material to
achieve specific goals
• Learn more about cellular processes,
including inheritance and gene
expression
• Provide better understanding and
treatment of diseases, particularly
genetic disorders
• Generate economic and social benefits
through production of valuable
biomolecules and improved plants and
animals for agriculture
Chapter5 13
Recombinant DNA

Genetic engineering utilizes


recombinant DNA technology
• Splicing together of genes or portions
of genes from different organisms

Recombinant DNA can be transferred


to plants and animals
• Modified animals are called transgenic
or genetically modified organisms (GMOs)
• Most modern biotechnology includes
manipulation of DNA
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Recombination in Nature
Many natural processes recombine
DNA:

Due to crossing over during


meiosis, each chromosome in a
gamete contains a mixture of
alleles from the two parental
chromosomes
• Thus, eggs and sperm contain
recombinant DNA
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Transformation

Bacteria can naturally take up


DNA from the environment
(transformation) and integrate
the new genes into the
genome (recombination)
Recombination DNA8
Chapter
Plasmid fragments13
in Bacteria transferr transferre
ed d
(a) Bacterium (b) to new(c) to new
host host

hromosome
Plasmid

Plasmid
1 µm replicates
in DNA
cytoplas fragment
m incorporat
ed into
chromoso
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Transformation

Small circular DNA molecules


(plasmids) carry supplementary
genes
• Plasmid genes may allow bacteria
to grow in novel environments
• Plasmid genes may enhance
virulence of bacteria in
establishing an infection
• Plasmid genes may confer
resistance to antimicrobial drugs
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Viral Transfer of DNA

Viral life cycle


1. Viral particle invades host cell
2. Viral DNA is replicated
3. Viral protein molecules are
synthesized
4. Offspring viruses are
assembled and break out of
the host cell
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Viral Transfer of DNA

Viral transfer of DNA


• Viruses may package some
genes from host cell into viral
particles during assembly
• Infection of new host cell
injects genes from previous
host, allowing for
recombination
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Viruses May Transfer Genes
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Biotechnology and Forensics

Forensics is the science of criminal


and victim identification

DNA technology has allowed forensic


science to identify victims and
criminals from trace biological
samples
Genetic sequences of any human
•Genetic
individual are unique
DNA analysis reveals patterns that
•DNA
identify people with a high degree of
accuracy
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Polymerase Chain Reaction

Forensic technicians typically


have very little DNA with
which to perform analyses
Polymerase Chain Reaction (PCR)
produces virtually unlimited
copies of a very small DNA
sample
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Polymerase Chain Reaction
PCR requires small pieces of DNA
(called primers) that are
complementary to the gene
sequences targeted for
copying
A PCR “run” is basically DNA
replication in a tiny test tube
• Template DNA, primer,
nucleotides, and DNA
polymerase are all in the
reaction mix
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Polymerase Chain Reaction
Four steps of a PCR cycle
1. Template strand separation
– The test tube is heated to 90-95oC to
cause the double stranded template
DNA to separate into single strands…

2. Binding of the primers


– The temperature is lowered to 50oC
to allow the primer DNA segments to
bind to the targeted gene sequences
through hydrogen bonding…
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Polymerase Chain Reaction

3. New DNA synthesis at targeted sequences


The temperature is raised to 70-72oC
where the heat-stable DNA polymerase
synthesizes new DNA of the sequences
targeted by the primers…

4. Repetition of the cycle


The cycle is repeated automatically (by a
thermocycler machine) for 20-30 cycles,
producing up to 1 billion copies of the
original targeted DNA sequence
Polymerase Chain Reaction: 1813
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(a) One PCR Cycle

90 °C 50 °C 72 °C

DNA Polymerase Primer


DNA

Original Separate Primers & DNA


Double- DNA DNA synthesize
helix Strands polymerase
DNA bind
Polymerase Chain Reaction: 1913
Chapter
(b) Multiple PCR Cycles

2 copies 4 copies 8 copies

DNA
ragment
to be
amplified
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Polymerase Chain Reaction

Choice of primers determines


which sequences are
amplified (copied)
Forensic scientists focus on
short tandem repeats (STRs)
found within the human
genome
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Polymerase Chain Reaction

STRs are repeated sequences of


DNA within the chromosomes
that do not code for proteins
STRs vary greatly between
different human individuals
A match of 10 different STRs
between suspect and crime
scene DNA virtually proves the
suspect was at the crime scene
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Gel Electrophoresis

Mixtures of DNA fragments can


be separated on the basis of
size
Gel electrophoresis is a technique
used to spread out different-
length DNA fragments in a
mixture
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Gel Electrophoresis
Four steps of gel electrophoresis
1. DNA mixtures are placed into wells
at one end of a slab of agarose gel

2. An electric current introduced


through the gel causes the
negatively-charged DNA
fragments to migrate towards the
positive electrode
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Gel Electrophoresis

Four steps of gel electrophoresis


3. Short DNA fragments move more easily
through the three-dimensional meshwork
of fibers between the gel
Short DNA fragments migrate farther than
long DNA fragments so the mixture is
separated into bands of DNA of specific
lengths
4. The invisible bands of DNA are made
visible using stains or DNA probes
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RFLP: Gel Electrophoresis

Larger
Direction fragments
of Migration move more
slowly;
smaller
fragments
move more
rapidly
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DNA Fingerprinting

DNA from a crime scene sample can be


amplified by PCR and run on a gel with
suspect DNAs
Short tandem repeats (STRs) in the gel
DNA can be identified by DNA probes
Distinctive pattern of STR numbers and
lengths are fairly unique to a specific
individual (forming a DNA fingerprint)
DNA fingerprint from crime scene can be
matched with DNA fingerprint of
suspect
DNA Fingerprint in 3013
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Forensics
: Which suspect A: #3 is prime
hould be suspect
dicted?

1 2 3 C  S 4 5 6 7
R  C
I E
SuspectsM NSuspects
E  E
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You can read the stuff on DNA


probes and Biotech in
Agriculture on your own…
Cut 3213
Restriction Enzymes Chapter
DNA

A DNA sequence (e.g. a gene)


can be removed from a
chromosome using special
enzymes

Restriction enzymes are nucleases


that cut DNA at specific
nucleotide sequences
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Cut 3413
Restriction Enzymes Chapter
DNA

Enzymes that create staggered


cuts with “sticky ends” are the
most useful in gene cloning
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Splicing of DNA Fragments

Sticky ends allow for splicing of


a DNA fragment with another
complementary fragment
• Bt gene can be cut out of the
Bacillus chromosome with the
same enzyme used to cut open
the plasmid
• Bt gene fragment ends can
base-pair with sticky ends of
the opened plasmid, adding
gene to the plasmid circle
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Splicing of DNA Fragments

DNA ligase enzyme used next to


permanently bond gene into
plasmid
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Plasmids Are Used to Insert
Genes

The Ti plasmid from


Agrobacterium tumefaciens is
ideal for transferring genes
into plant chromosomes
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Plasmids Are Used to Insert
Genes

Agrobacterium infects plant


cells and inserts its small Ti
plasmid into a plant
chromosome in the nucleus
• Pathogenic effects of certain
tumor-causing Ti plasmid genes
can be disabled
• A gene inserted into a Ti
plasmid is therefore carried into
the plant cell chromosomes by a
natural process
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The Human Genome Project

Findings
• Human genome contains ~25,000 genes
• New genes, including many disease-
associated genes have been discovered
• Has determined the nucleotide sequence
of all the DNA in our entire set of genes,
called the human genome
• The genes comprise 2% of all the DNA
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The Human Genome Project

Applications
• Improved diagnosis, treatment
and cures of genetic disorders or
predispositions
• Comparison of our genome to
those of other species will clarify
the genetic differences that help
to make us human
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DNA Probes

Defective alleles can also be


identified using DNA probes
DNA probing is especially useful
where there are many
different alleles at a single
gene locus
• Cystic fibrosis is a disease
caused by any of 32 alleles out
of 1000 total possible alleles
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DNA Probes

Arrays of single-stranded DNA


complementary to each of the
defective alleles can be bound to
filter paper
A person’s DNA sample is cut up and
separated into single-strands
The array is bathed in the DNA sample
Strands of DNA binding to complementary
sequence on the paper indicate
presence of a defective allele in
person’s genome
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DNA Probes

An expanded version of this type of


DNA analysis is known as a
microarray
A microarray contains up to
thousands of probes for a variety
of disease-related alleles
Microarray analysis has the potential
to comprehensively identify
disease susceptibility
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to 5313
Scientific Objections Chapter
GMOs

Safety issues from eating GMOs


• Could ingestion of Bt protein in insect-
resistant plants be dangerous to humans?
• Are transgenic fish producing extra growth
hormone dangerous to eat?
• Could GM crops cause allergic reactions?
– USDA now monitors GM foods for allergic
potential
• Toxicology study of GM plants (2003)
concluded that ingestion of current
transgenic crops pose no significant health
dangers
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Scientific Objections Chapter
GMOs

Environmental hazards posed by GMOs


• Pollen from modified plants can carry GM
genes to the wild plant population
– Could herbicide resistance genes be
transferred to weed species, creating
superweeds?

• Could GM fish reduce biodiversity in the


wild population if they escape?
– Reduced diversity in wild fish makes them
more susceptible to catastrophic disease
outbreaks
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Scientific Objections Chapter
GMOs

Environmental hazards posed


by GMOs
• US found to lack adequate
system to monitor changes in
ecosystem wrought by GMOs
(National Academy of Science
Study 2003)
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The Human Genome

Should parents be given information about the


genetic health of an unborn fetus?

Should parents be allowed to select the


genomes of their offspring?
• Embryos from in vitro fertilization are currently
tested before implantation
• Many unused embryos are discarded

Should parents be allowed to design or correct


the genomes of their offspring?
Hope through Gene 5713
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Therapy
Human Cloning:
Permanent Genetic 5813
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Correction?
Parents with Baby with
Zygote withgenetic
genetic disease defective gene disord

Viral
vector
with
herapeutic
gene Cell Culture Embryo with
defective gene
Treated Culture Genetically
corrected
Enucleated embryo clone
Genetically
egg cellcorrected
egg cell
netically corrected
enetically
cell from culture Healthy baby
Chapter 13

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