Download Medical and Ethical Implications of Inexpensive Genome

The Race for the $1000 Genome:
Medical and Ethical Implications of Inexpensive Genome Sequencing
Kathryn Fluss, Juyoung Guag, Jeff Kleinberg, Bipin Rajendran
Introduction
Basic nucleic acid sequencing methods primarily rely on three steps; these
steps include cleaving the genome into shorter, more manageable
sequences, sequencing these smaller segments and then determining the
order in which these sequences make up the complete genome. The
evolution of genome sequencing has occurred rapidly over the course of
about 30 years as companies strive to create the “$1000 genome” a price
many consider affordable enough for everyday use. The use of
inexpensive genome sequencing, however, has generated widespread
controversy over the ethical issues associated with its use in the medical
setting.
Chain-Terminator Method
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Automated Sequencing
Developed by Frederick Sanger in 1974, the Chain Terminator (dideoxy) method is the most commonly used
sequencing method.
Four reaction mixtures are prepared for gel electrophoresis; each mixture contains the single-stranded template
strand, primer, four dNTPs, and one of four ddNTPs. Each mixture is loaded into one of four lanes (C,G,A,T).
After electrophoresis, sequence is read from bottom to top; this sequence is the complementary strand, and the
sequence of the template can be determined
Requires gel with sufficient resolving power (the ability to distinguish between chains that differ by a single
nucleotide.)
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Automated fluorescent dye-terminator cycle sequencing was developed by Leroy Hood in 1986
Uses the same principle of dideoxynucleotide chain termination that is applied in the Sanger Method
dNTP’s or ddNTP’s are added in the 5’ to 3’ direction; when the ddNTP is incorporated, the extension
stops.
All four bases show a different fluorescence which emits a unique wavelength.
All four reactions can run in a single lane
A laser detector detect emission of each wavelength from bottom and determine bases and the gene
sequence is confirmed in both directions.
Snake Venom Phosphodiesterase
• Used by Robert Holley in 1965 to sequence 76-residue tRNA molecule
• Removes one nucleotide at a time, starting at the 3’ end, by breaking the
phosphodiester bond.
The lack of a 3’OH
group on a dNTP
signals the termination
of chain growth as
there is no free 3’OH
for another nucleotide
to attach
New Techniques: Sequencing by Synthesis
•Real time detection without electrophoresis or labeled primers.
•Based on the detection of pyrophosphate that is released when a nucleotide is added.
• Produces a set of fragments of all possible lengths.
• The size of each fragment can be determined with mass spectrometry.
• Since each fragment only differs from the fragment before it by one nucleotide, the
fragments can be compared to identify the nucleotides at the 3’ ends.
• Analysis is time consuming.
• Can be used to characterize modified oligonucleotides.
"The sequence information provides a starting point
from which the real research into the thousands of
diseases that have a genetic basis can begin. The
sooner we can get to this starting point, the sooner we
can begin to see a payoff in ultimately improving
human health.”
sulfurylase
PPi
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Developed by J. Craig Venter in 1996
Major principle is that it is random sampling; 500-700
nucleotides are read and then assembled to construct whole
genome sequence
Clone is randomly sheared into small fragment (around 1Kb)
Subcloned into a cloning vector
These serve as primers during PCR
Amplified fragment assemble by overlapping
- J. Craig Venter
454 Life Sciences Corp. Method
"We used to think that our fate was in our stars. Now we know that, in large measure, our fate is in our genes."
Francis Crick
The Race to the $1000 Dollar Genome
Company
Format
Read Length
(bases)
Expected
Throughput
Mb (million
bases)/day
Parallel bead array
100
96
Sequencing by ligation
50
200
Applied Biosystems
Capillary electrophoresis
1000
3-4
LI-COR Biosciences
Electronic microchip
20,000
14,000
Microchip Biotechnologies
Parallel bead array
850-1000
7
Network Biosystems
Biochip
800+
5
NimbleGen Systems
Map and survey microarray
30
100
Parallel microchip
35
500
Single-molecule array
NA
1000
454 Life Sciences
Agencourt Bioscience
Solexa
Medical Implications of Genomic Sequencing
Early Detection of Abnormal Genes
- Cystic Fibrosis is caused by a mutation in the CFTR (cystic fibrosis transmembrane
conductance regulator) gene
- A single mutated gene causes other diseases such as Huntington’s disease and
Duchenne muscular dystrophy, and those genes have already been identified.
- Mutations in many genes believed to be a major risk factor for Alzheimer’s disease.
So far the only one mutation for late-onset Alzheimer’s is at epsilon-4 position of
apolipoprotein E (APoE) gene.
• People who were found with genetic disorders could alter their lifestyle to
prevent disease or reduce its severity.
- For example, recent studies show regular exercise helps reduce the risk of
Alzheimer’s disease onset.
• Directions of Future Medical Research
- Genomic sequencing will be a major target for medical research for the next few
decades.
- Identifying a genetic link to more diseases, especially those that are caused by multiple
abnormal genes.
- Is the varying efficiency and side effects of drugs for each individual related to their
genes?
- More easily chose the best drugs for everyone
- People with mutant MTHFR (methylenetetrahydrofolate reductase) may have
more severe side effects to antifolate chemotherapy.
Light (Detection indicates
nucleotide addition)
ATP
•Used in an automated system developed by 454 Life Sciences Corp. that does not require
cloning into bacterial vectors.
Shotgun Sequencing
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luciferase
VisiGen Biotechnologies
A list of companies involved in the quest to create the $1000 genome sequence. Such technology must demonstrate low cost, high accuracy,
the ability to read long stretches of DNA and high throughput (Source: http://www.sciencemag.org/cgi/content/full/311/5767/1544#table).
Acknowledgements
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Dr. Jason Kahn
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http://www.454.com/
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http://www.bio.davidson.edu/courses/genomics/method/shotgun.html
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http://www.clinical-virology.org/pages/cvn/vir_all/cvn_gp_how.html
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http://medstat.med.utah.edu/block2/biochem/Formosa/Figures/Lecture6/6-08%20Sequencing%20Modern.GIF
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http://www.cnn.com/SPECIALS/2000/genome/story/medical.implications
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http://www.nia.nih.gov/Alzheimers/Publications/geneticsfs.htm
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http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/cf.shtm
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http://www.sciencemag.org/cgi/content/full/311/5767/1544#table
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http://www.uvm.edu/~cgep/Education/Sequence.html
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Margulies, M., et al. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 437:376-80.
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Ronaghi, M., S. Katamohamed, B. Pettersson, M. Uhlen, P. Nyren. 1996. Real-time DNA sequencing using detection of
pyrophosphate release. Anal. Biochem. 242:84-89.
Voet, D., J. Voet, C.W. Pratt. Fundamentals of Biochemistry. New York: Wiley, c2002.
Wu, H. and H. Aboleneen. 2000. Sequencing oligonucleotides with blocked termini using exonuclease digestion and
electrospray mass spectrometry. Anal. Biochem. 287:126-135.
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•Shear the genome,
ligate fragments to
adaptors, and bind
individual fragments
to beads.
•Perform PCR on an
emulsion of the PCR mix
in oil. This will allow
each fragment to be
amplified individually
without isolating them
from the mixture.
•Total sequence is assembled
by combining overlapping
fragments (similar to shotgun
sequencing).
•Beads are deposited into wells
along with other reagents.
•Nucleotides flow
past the wells,
and incorporation
is measured
based on the
generation of
photons.
Ethical Implications of Genome Sequencing
• Health vs. Superiority?
With the introduction of gene therapy, a new form of treatment has risen. But what
happens when gene therapy shifts from treating disorders to making a healthy
individual stronger, faster or smarter?
• Availability?
Would genome sequencing and therapy become available to everyone, rich or poor?
Researchers are working towards developing the “$1000 genome,” a reasonable price
that would make genomic sequencing available to many.
• Insurance?
With the advent of genome sequencing, insurance companies could possibly use this
technology to screen individuals before granting them coverage. If such a test reveals
an individual is predisposed to a certain disorder, does an insurance company have the
right to deny coverage?
• Limited opportunities?
Employers and educational institutions, much like insurance companies, could
potentially deny access to individuals based on information found in their genomes.
Should such information even be allowed for viewing by these institutions?