Download MB206_fhs_int_013b_ST_Jan09

By
Amani Ahmad
Khoo Lee Ting
Heike Priebe
Aida May
16 March 2009
Running the reaction of all
the dideoxy nucleotides
using different dyes
generates this type of
diagram in same lane.
• Sometimes the spacing
between the bands is hard to
measure.
• Thus use machine to run and
read the electrophoresis.
• Capillary electrophoresis: the
fragments are piped through a
tiny glass-fiber capillary
during the electrophoresis
step, and they come out the far
end in size-order.
 Chemical cleave method
 Sequence small fragments of
DNA
 The radioactive labelling is
done on the dsDNA.
 Division of aliquots is done
by methylation or removal
of base.
 Requires DNA
 Breaks DNA at different
nucleotides
 Enzymatic cleave method
 Sequencing small fragments are
problematic.
 The radioactive labelling is done
on the ssDNA.
 Allow high throughput
automated sequencing
techniques.
 Allow Real Time detection.
 Requires DNA synthesis
 Termination of chain elongation
Wilson and Walker, 224
Conclusion
• Reasons for DNA Sequencing
– Philosophical reasons
– Pioneering reasons
– Practical day-to-day reasons
•
•
•
•
•
Cost
Legality of DNA Sequencing
Comparison of the DNA Sequencing Methods
Genetically Engineered Products
Genetics in the Works
Reasons for DNA Sequencing
• Philosophical reasons :
– Deciphering “code of life”
• Pioneering reasons :
– Understanding organisms, physiology, evolution,
disease, cellular behaviour, etc.
• Practical day-to-day reasons :
–
–
–
–
checking mutations
checking constructs in cloning
constructing phylogenies
finding genes
Cost
Cost is dependant on a number of factors but
typically in 2003:
• Each tube of sample DNA costs $27 to run.
• An entire set of 96 tubes from one source (the
capacity of the present equipment) costs $960.
• The methods used will readily analyze DNA
fragments of 500-1000 bases in length,
depending on the quality of DNA used
* The dye alone to run 5000 reactions costs $61,000
Legality of DNA Sequencing
• Should gene therapy and cloning be regulated
by the government?
• What would happen if genes being inserted into
a patient went to the wrong chromosome?
• If plants and animals are altered, will the
balance of nature be disrupted? Will "designer"
babies be created?
• What do you call your mother if she's your clone,
and therefore also your twin sister?
Comparison of the DNA
Sequencing Methods
Maxam-Gilbert Method
Sanger Method
1
Manual sequencing
2
More effort needed in
running gels
Automatic sequencing
(Chain-termination
method)
Uses automated technology
3
Time consuming
Easier, faster (1 lane per
sample instead of 4)
4
Radioactivity is used to label
the fragments
Uses special fluorescent dyes
to label the DNA
fragments
Genetically Engineered Products
Product
Alpha-interferon
Usage
 Used to shrink tumour
 Used to treat hepatitis B and C
Beta-interferon
 Treats multiple sclerosis
Humulin (human
insulin)
Monoclonal
antibodies
 Treats diabetes
 Works against cancerous cells
 An option for chemotherapy
Tissue plasminogen  Prevents blood clots
activator (tPA)
 Prevents heart attacks or stokes
Genetics in the Works
Product/Research Expected Effect
Area
Functional
 Function of DNA sequences of
genomics
an organism is studied
Microarray analysis  Thousands of genes can be
studied at one time or in many
different organisms at once.
Antisense therapy  Research done to stop the
function of bad genes
Creating new
 Cure for diseases
chromosomes
Reference
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•
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CGDP - DNA Sequencing Part 1, FLMNH © 2005
[Online} Available from:
<http://www.flmnh.ufl.edu/cowries/sequence3.html>
[Accessed 4th March 2009]
Exploring Genetics Research; Genetic pioneering.
[Online] Available from:
<http://www.dummies.com/how-to/content/exploringgenetics-research.htmll> [Accessed 4th March 2009]
DNA Sequencing Technologies By: Jill U. Adams,
Ph.D © 2008 Nature Education. [Online] Available
from: < http://www.nature.com/scitable/topicpage/DNASequencing-Technologies-690 > [Accessed 4th March
2009]
• Watson J D, Gilman M, Witkowski J and
Zoller M, Recombinant DNA, second
edition, 1992, scientific American Inc.
• Wilson K and Walker J, Principles and
Techniques of Biochemistry and Molecular
Biology, sixth edition, 2005, Cambridge
University Press
The End
Thank you