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Biotechnology
2007-2008
A Brave New World
Practical DNA Technology Uses
• Forensics
– Sequencing DNA of crime suspects
• Diagnosis of disease
– DNA screening for diseases
• Human gene therapy
– Replacing absent or faulty DNA with normal,
working DNA
• Pharmaceutical products (vaccines)
• Transgenic organisms
– Animals
• Mice with human genes for animal testing
• Livestock with extra copies of growth hormone
genes to improve food supply
• Chicken with a gene resistant to the bacteria
that causes food poisoning
– Plants
• 52% of soybeans and 25% of corn in U.S. are
transgenic.
Human Genome Project
Mapping the Human Genome
•
•
•
•
Complete in 2003
46 chromosomes
3.2 billion DNA base pairs
19,599 protein-coding
sections
– Genes make up 2% of Human
DNA
• 98% of DNA is non-coding
– Entire function is still unknown,
but does play a role in gene
regulation
Cloning
• Cloning is a term that
refers to making a
genetically identical
copy.
• Cells and Tissues can
be cloned.
• Organism cloning
(also called
reproductive cloning)
refers to making a
new multicellular
organism, genetically
identical to another.
How to Clone
Take a donor’s egg and remove the nucleus.
Insert a nucleus from the targeted individual’s diploid cell.
Dolly
• In 1996, Ian Wilmut
cloned Dolly from an
adult sheep.
CopyCat
• In February 2002,
researchers from Texas A
& M reported the live
birth of a cloned tabby.
• Researchers are
interested in using
cloned cats in AIDS
research, since feline
AIDS is a good model for
human AIDS.
Noah (Ox Cloned in a Cow) in
2001
PCR
• Polymerase Chain
Reaction
– method for making
many, many copies
of a specific
segment of DNA
– ~only need 1 cell of
DNA to start
PCR process
• In tube: DNA, DNA polymerase, primer, nucleotides
• Denature DNA: heat (90°C) DNA to separate strands
• Anneal DNA: cool to hybridize with primers & build DNA
(extension)
What does 900C
do to our
DNA polymerase?
The polymerase problem
• 90°C heat destroys DNA polymerase
• Need enzyme that can
withstand 90°C…
– Taq polymerase (Thermus aquaticus)
• from hot springs bacteria
Sequencing DNA
• A PCR is run four times; each
time with a different
dideoxynucleotide, or
“ddNTP,” which lack the 3'hydroxyl group.
• Whenever a ddNTP is
incorporated into a growing
DNA chain, it stops chain
growth.
• The resulting pieces can be
analyzed for length to
determine the sequence of
the DNA.
Restriction enzymes
• Cut DNA at specific sites
– leave “sticky ends”
restriction enzyme cut site
GTAACGAATTCACGCTT
CATTGCTTAAGTGCGAA
restriction enzyme cut site
GTAACG AATTCACGCTT
CATTGCTTAA GTGCGAA
Sticky ends
• Cut other DNA with same enzymes
– leave “sticky ends” on both
– can glue DNA together at “sticky ends”
GTAACG AATTCACGCTT
CATTGCTTAA GTGCGAA
gene
you want
GGACCTG AATTCCGGATA
CCTGGACTTAA GGCCTAT
chromosome
want to add
gene to
GGACCTG AATTCACGCTT
CCTGGACTTAA GTGCGAA
combined
DNA
Process of DNA Fingerprinting
• First restriction enzymes cut the DNA into
fragments
• The DNA fragments are separated
fragments by size
with Gel Electrophoresis
Gel electrophoresis
• DNA is negatively charged
• When it’s in an electrical field
it moves toward the positive
side
• small pieces travel farther
• large pieces travel slower
DNA
–

“swimming through Jello”
+
Gel Electrophoresis
DNA &
restriction enzyme
longer fragments
wells
power
source
gel
shorter fragments
+
completed gel
Uses: Evolutionary relationships
• Comparing DNA samples from different
organisms to measure evolutionary
relationships
turtle snake rat squirrel
–
DNA

+
1
2
3
4
5
1
2
3
4
fruitfly
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
+
Pre-Implantation Genetic Diagnosis (PGD)
Removing a cell for
diagnosis from a human
embryo.
Amniocentesis and Chorionic Villus
Sampling
Many new
techniques for
learning about
individual genes
rather than whole
chromosomes are
available or
under
development.
Uses: Paternity
• Who’s the father?
–
DNA

+
Mom
F1
F2
child
Uses: Forensics
• Comparing DNA sample from crime scene
with suspects & victim
suspects
S1 S2 S3
crime
scene
V sample
–DNA

+
Forensic DNA fingerprinting
• Any two humans have 99.9% similar DNA
•0.1% of 3 billion base pairs is still 30 million.
•The protein-coding sections of our DNA may
lose their function if sections are repeated.
•However, the non-coding sections of DNA can
contain several short (2-4 bases) repeats called
Short Tandem Repeats (STR).
•The number of repeats changes the length of
DNA.
Differences at the DNA level
• Two sections of “junk” DNA
3 Repeats
GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
GCTTGTAACGGCATCATCATCATCATCATCCGGCCTACGCTT
CGAACATTGCCGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA
6 Repeats
DNA patterns for DNA fingerprints
Allele 1
cut sites
repeats
cut sites
GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
Cut the DNA
GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTT
CGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA
1
2
– DNA 
allele 1
3
+
Differences between people
Allele 1
cut sites
cut sites
GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA
Allele 2: more repeats
GCTTGTAACGGCCTCATCATCATCATCATCATCCGGCCTACGCTT
CGAACATTGCCGGAGTAGTAGTAGTAGTAGTAGGCCGGATGCGAA
1
2
DNA fingerprint
– DNA 
allele 1
allele 2
3
+
RFLPs
• Restriction Fragment Length Polymorphism
– differences in DNA between individuals
Alec Jeffries
1984


change in DNA sequence affects restriction enzyme
“cut” site
creates different fragment sizes & different band
pattern
RFLP / electrophoresis use in forensics
• 1st case successfully using DNA evidence
– 1987 rape case convicting Tommie Lee Andrews
“standard”
semen sample from rapist
blood sample from suspect
“standard”
How can you
compare DNA from
blood & from semen?
RBC?
“standard”
semen sample from rapist
blood sample from suspect
“standard”
Electrophoresis use in forensics
• Evidence from murder trial
– Do you think suspect is guilty?
blood sample 1 from crime scene
blood sample 2 from crime scene
blood sample 3 from crime scene
“standard”
blood sample from suspect
OJ Simpson
blood sample from victim 1
N Brown
blood sample from victim 2
R Goldman
“standard”
Bacterial genome
• Single circular chromosome
–haploid
–~4 million base pairs
• 1/1000 DNA in eukaryote
How have these
little guys gotten to
be so diverse??
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!!
– can be imported from
environment
Plasmids help us get new genes into
bacteria
• Insert new gene into plasmid
• Insert plasmid into bacteria = vector
• bacteria now expresses new gene and makes
new protein
transformed
gene from
other organism
recombinant
plasmid
cut DNA
+
vector
glue DNA
plasmid
DNA
 RNA  protein  trait
bacteria
Grow bacteria…make more
transformed
gene from
other organism
bacteria
recombinant
plasmid
+
vector
plasmid
grow
bacteria
harvest (purify)
protein
Plasmids are also used to genetically
engineer multicellular organisms
• Plasmids are DNA vectors
• Genes must be inserted into the zygote to change
the traits of a multicellular organisms.
• DNA combined from different sources is called
Recombinant DNA
• An organism with Recombinant DNA is called
Transgenic
p-GLO Gene
• The gene that makes jellyfish glow has been
isolated and inserted into other organisms.
Uses of genetic engineering
• Genetically modified organisms (GMO)
– 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
Gene Therapy
•
Problem (disease-causing) genes can be
removed and replaced with normal genes.