DNA Sequencing

Overview of Sequencing
• Most powerful methods ever devised for
DNA analysis
• It is used to:
– Determine the precise order of base sequence
– Determine all restriction sites within a gene
– Predict protein product
– Provide information about potential sites of
introns in genome
– Gives information about mechanism of
expression of a gene
 Two Methods

Chemical Cleavage Enzymatic Cleavage

Maxam-Gilbert Sanger
Chemical Cleavage
Maxam-Gilbert

1970, based on chemical cleavage of restriction

fragments.
The fragments are end-labeled with radioactive
nuclei.
The resulting sub fragments are separated by gel
electrophoresis and labeled fragments are
detected by autoradiography.
Walter Gilbert Fredrick sanger
Mary-Clair King- BRCA 1
Enzymatic Cleavage
Sanger

Based on random termination

of a DNA chain during
enzymatic synthesis.
The technique is possible because the dideoxy
analogue of each of the four normal
nucleotides can be incorporated into a growing
chain by DNA polymerase.
 Cell-Based Cloning (1)

Case: A thirteen year old girl is admitted with

dehydration, vomiting & weight loss. Her blood
glucose level is 344mg/dL (19.1mmol/L) and she
has ketonuria. A diagnosis of type 1 diabetes
mellitus. She is started on recombinant human
insulin, is rehydrated, and makes a prompt
recovery.
Cell-Based Cloning (2)
•Previously insulin therapy involved the use of animal
insulins, most commonly pork or beef, which were chemically
similar, but not identical, to human insulin.
•Animal insulins often led to the development of anitbodies,
which reduced the efficacy of the insulin and could lead to
treatment failures.
•Following the cloning of the human insulin gene, large scale
production of pure human insulin was performed by inserting
the cloned gene into an invitro amplification system.
•Thus, large amounts of insulin gene copies may be produced,
which are then expressed in either bacteria or yeast and the
resulting purified insulin is then available for use in diabetic
patients.
Automated Gene Sequencing
• The human genome project’s aim is to obtain the
DNA sequence of the entire human genome.
• By product of this project was the development of
technology such as automation of the sequencing
process.
• Automated DNA sequencing uses fluorescent rather
than radio-labeled products.
• Using four different fluorescent tags, one for each
sequencing reaction, resulted in
– All four reactions can run on the same lane
– Scanner device can then read the gel
– Enables four times as many reactions to be performed for gel
– Autoradiography time is abolished.
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