Running the reaction of all the dideoxy

nucleotides using different dyes

generates this type of diagram in same lane.

• Sometimes the spacing Sometimes the spacing between the bands is between the bands is hard to measure.hard to measure.

• Thus use machine to run Thus use machine to run and read the and read the electrophoresis.electrophoresis.

• Capillary electrophoresis:Capillary electrophoresis: the fragments are piped the fragments are piped through a tiny glass-fiber through a tiny glass-fiber capillary during the capillary during the electrophoresis step, and electrophoresis step, and they come out the far they come out the far end in size-order.end in size-order.

Chemical cleave methodChemical cleave method Sequence small Sequence small

fragments of DNAfragments of DNA The radioactive labelling The radioactive labelling

is done on the dsDNA.is done on the dsDNA. Division of aliquots is Division of aliquots is

done by methylation or done by methylation or removal of base.removal of base.

Requires DNARequires DNA Breaks DNA at different Breaks DNA at different

nucleotidesnucleotides

Enzymatic cleave methodEnzymatic cleave method Sequencing small Sequencing small

fragments are fragments are problematic.problematic.

The radioactive labelling The radioactive labelling is done on the ssDNA.is done on the ssDNA.

Allow high throughput Allow high throughput automated sequencing automated sequencing techniques. techniques.

Allow Real Time detection. Allow Real Time detection. Requires DNA synthesisRequires DNA synthesis Termination of chain Termination of chain

elongationelongation

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

CostCost 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?

Maxam-Gilbert Method Sanger Method

Manual sequencing Automatic sequencing (Chain-termination method)

More effort needed in running gels

Uses automated technology

Time consuming Easier, faster (1 lane per sample instead of 4)

Radioactivity is used to label the fragments

Uses special fluorescent dyes to label the DNA fragments

Comparison of the DNA Sequencing Methods

1

2

3

4

Genetically Engineered Products

Product Usage

Alpha-interferon Used to shrink tumour Used to treat hepatitis B and C

Beta-interferon Treats multiple sclerosis

Humulin (human insulin)

Treats diabetes

Monoclonal antibodies

Works against cancerous cells An option for chemotherapy

Tissue plasminogen activator (tPA)

Prevents blood clots Prevents heart attacks or stokes

Genetics in the Works

Product/Research Area

Expected Effect

Functional genomics

Function of DNA sequences of 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 chromosomes

Cure for diseases

Reference

• 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/exploring-genetics-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/DNA-Sequencing-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