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Biotechnology Tools
DNA Sequencing
DNA Sequencing
– dideoxynucleotides
• ddATP, ddGTP, ddTTP,
ddCTP
• missing O for bonding of
next
nucleotide
• terminates the growing
chain
DNA Sequencing
• Sanger method
– synthesize
complementary DNA
strand in vitro
– in each tube:
• “normal” N-bases
• dideoxy N-bases
–ddA, ddC, ddG,
ddT
• DNA polymerase
2
• primer
• buffers & salt
1
2
3
4
Reading the sequence
• Load gel with sequences from ddA, ddT,
ddC, ddG in separate lanes
– read lanes manually & carefully
– polyacrylamide gel
Fred Sanger
• This was his 2nd Nobel Prize!!
– 1st was in 1958 for the structure
of insulin
1978 | 1980
The Sanger Method:
DNAi video
tutorial
Advancements in sequencing
• Fluorescent tagging
– no more radioactivity
– all 4 bases in 1 lane
• each base a different color
• Automated reading
More Advancements in
sequencing
 Capillary tube electrophoresis
no more pouring gels
higher capacity & faster
Applied Biosystems, Inc
(ABI) built an industry on
these machines
 Big labs!
economy of scale
PUBLIC
 Joint Genome Institute
(DOE)
 MIT
 Washington University of
St. Louis
 Baylor College of
Medicine
 Sanger Center (UK)
PRIVATE
 Celera Genomics
Automated Sequencing
machines
 Really BIG labs!
Human Genome Project
• U.S government project
– begun in 1990
• estimated to be a 15 year
project
– DOE & NIH
• initiated by Jim Watson
• led by Francis Collins
– goal was to sequence entire
human genome
• 3 billion base pairs
• Celera Genomics
– Craig Venter challenged gov’t
– would do it faster, cheaper
– private company
Different approaches
gov’t method
Craig Venter’s method
“map-based method”
“shotgun method”
1. Cut DNA segment into fragments,
arrange based on overlapping
nucleotide sequences, and clone
fragments.
2. Cut and clone into smaller fragments.
3. Assemble DNA sequence
using overlapping sequences.
1. Cut DNA entire chromosome
into small fragments and clone.
2. Sequence each segment &
arrange based on overlapping
nucleotide sequences.
Shotgun!
Human Genome Project
• On June 26, 2001, HGP published the “working draft”
of the DNA sequence of the human genome (4 years
ahead of schedule).
• Historic Event!
– blueprint
of a human
– the potential to
change science &
medicine
Sequence of
46 Human Chromosomes
3G of data
3 billion base pairs
Things Are Strange In Here:
Raw genome data
NCBI GenBank
• Database of
genetic
sequences
gathered
from
research
• Publicly
available on
Web!
Maps of human genes…
• Where the genes are…
– mapping genes & their mutant alleles
Defining a gene…
• “Defining a gene is problematic because…
one gene can code for several protein
products, some genes code only for RNA, two
genes can overlap, and there are many other
complications.”
•
– Elizabeth Pennisi, Science 2003
protein
gene
gene
RNA
polypeptide 1
gene
polypeptide 2
polypeptide 3
Types of DNA sequences in the
human genome
Exons (regions of genes coding
for protein, rRNA, tRNA) (1.5%)
Repetitive
DNA that
includes
transposable
elements
and related
sequences
(44%)
Introns and
regulatory
sequences
(24%)
Repetitive
DNA
unrelated to
transposable
elements
(about 15%)
Alu elements
(10%)
Simple sequence
DNA (3%)
Large-segment
duplications (5–6%)
Unique
noncoding
DNA (15%)
And we didn’t stop there…
Genome Sizes and Estimated Numbers of Genes*
What have we found?
• When you go looking…
…you will certainly find
something!
Sing A Silly Song Extolling
Automation!
Ethical Questions…
•
•
1. You have a familiar history of a terminal genetic
disease. A genetic test exists for the disease.
Would you take the test to see if you will have the
disease?
•
2. As a condition of your continued employment,
your boss wants you to have a genetic screening
test and the results sent to the company. Will you
get the test?
3. You and your spouse have decided to have a
child. You have the resources to pre-determine
your child’s gender. Would you do this?
4. What about your child’s intelligence?
•
5. You discover your favorite food contains
genetically modified ingredients. Will you
continue to eat the food?
6. As part of a routine medical procedure, your
doctor discovers that you have a rare, beneficial
variant of a protein that protects you from heart
disease. Should your doctor be able to patent the
protein?
7. Should you be entitled to any money from the
patent?