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A Lot More Advanced Biotechnology Tools. DNA Sequencing. DNA Sequencing. dideoxynucleotides ddATP, ddGTP, ddTTP, ddCTP missing O for bonding of next nucleotide terminates the growing chain. 2. 1. 3. 4. 2. DNA Sequencing. Sanger method synthesize complementary DNA strand in vitro - PowerPoint PPT Presentation
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A Lot More Advanced Biotechnology Tools
DNA Sequencing
– dideoxynucleotides• ddATP, ddGTP, ddTTP,
ddCTP• missing O for bonding of
nextnucleotide
• terminates the growing chain
DNA Sequencing
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• primer• buffers & salt
2
1
3
4
2
Reading the sequence• Load gel with sequences from ddA,
ddT, ddC, ddG in separate lanes– read lanes manually & carefully– polyacrylamide gel
Fred Sanger 1978 | 1980
• This was his 2nd Nobel Prize!!– 1st was in 1958 for the
structure of insulin
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
Applied Biosystems, Inc Applied Biosystems, Inc (ABI) built an industry on (ABI) built an industry on these machinesthese machines
More Advancements in sequencing
Capillary tube electrophoresisno more pouring gelshigher capacity & faster
PUBLICPUBLICJoint Genome Institute Joint Genome Institute
(DOE)(DOE)MITMITWashington University of Washington University of
St. LouisSt. LouisBaylor College of Baylor College of
MedicineMedicineSanger Center (UK)Sanger Center (UK)PRIVATEPRIVATECelera GenomicsCelera Genomics
Big labs!economy of scale
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
3. Assemble DNA sequence using overlapping sequences.
““map-based method”map-based method”gov’t methodgov’t method
““shotgun method”shotgun method”Craig Venter’s methodCraig Venter’s method
1. Cut DNA entire chromosome 1. Cut DNA entire chromosome into small fragments and clone.into small fragments and clone.
2. Sequence each segment & 2. Sequence each segment & arrange based on overlapping arrange based on overlapping nucleotide sequences.nucleotide sequences.
1.1. Cut DNA segment into fragments, Cut DNA segment into fragments, arrange based on overlapping arrange based on overlapping nucleotide sequences, and clone nucleotide sequences, and clone fragments.fragments.
2. Cut and clone into smaller fragments.2. Cut and clone into smaller fragments.
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
3 billion base pairs
3G of data
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
gene
polypeptide 1polypeptide 1
polypeptide 2polypeptide 2
polypeptide 3polypeptide 3
proteinproteingene
RNARNAgene
Types of DNA sequences in the human genome Exons (regions of genes coding
for protein, rRNA, tRNA) (1.5%)
RepetitiveDNA thatincludes
transposableelements
and relatedsequences
(44%)
Introns andregulatorysequences
(24%)
UniquenoncodingDNA (15%)
RepetitiveDNA
unrelated totransposable
elements(about 15%)
Alu elements(10%)
Simple sequenceDNA (3%)
Large-segmentduplications (5–6%)
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?