1. EVALUATION SEMINAR
ON
DNA SEQUENCING
PRESENTED BY
AMIT SHRESTHA
M-PHARM
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2. DNA
Deoxyribonucleic acid (DNA) is a nucleic acid that
functions include
 Storage of genetic information
 Self-duplication & inheritance.
Expression of the genetic message.
DNA’s major function is to code for proteins.
Information is encoded in the order of the nitrogenous
bases.
Along with RNA and proteins, DNA is one of the three major
macromolecules essential for all known forms of life.
Most DNA molecules are double-stranded helices, consisting
of two long biopolymers of simpler units called
nucleotides—each nucleotide is composed of a
nucleobase (guanine, adenine, thymine, and cytosine),
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3. Watson & Crick Model
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4. DNA SEQUENCING
Determining the order of bases in a section
of DNA
four bases—adenine
guanine
cytosine
thymine
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5. PURPOSE
• Deciphering “code of life”
• Detecting mutations
• Typing microorganisms
• Identifying human halotypes
• Designating polymorphisms
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6. DNA SEQUENCING METHODS
Historically there are two main methods of
DNA sequencing:
1.Maxam and Gilbert method
2.Sanger method
Modern sequencing equipment uses the
principles of the Sanger technique.
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7. MAXAM & GILBERT METHOD
• A. M. Maxam and W.Gilbert-1977
• The sequence of a double-stranded or
single-stranded DNA molecule is
determined by treatment with
chemicals that cut the molecule at
specific nucleotide positions.
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8. PRINCIPLE :
Chemical degradation
Reaction in two stages:
• Chemical modification of the bases
• Modified base is removed from its
sugar, pyperidin cleaves phosphodiester bonds
5’ and 3’ and base is released
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9. The method requires radioactive labelling at one end and
purification of the DNA fragment to be sequenced.
Chemical treatment generates breaks at a small proportion of one
or two of the four nucleotide bases in each of four reactions
(G, A+G, C, C+T).
Thus a series of labelled fragments is generated, from the
radiolabelled end to the first 'cut' site in each molecule.
The fragments in the four reactions are arranged side by side in geL
electrophoresis for size separation.
To visualize the fragments, the gel is exposed to X-ray film for
autoradiography, yielding a series of dark bands each
corresponding to a radiolabelled DNA fragment, from which the
sequence may be inferred.
PROCEDURE
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11. SANGER METHOD
• Most common approach used to
sequencing DNA.
• Invented by Frederick Sanger - 1977
• Nobel prize - 1980
• Also termed as chain termination or
dideoxy method
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12. SANGER METHOD TERMED AS
CHAIN TERMINATION METHOD
This method uses dideoxynucleotide triphosphates
(ddNTPs) chain terminators :
which have an H on the 3’ carbon of the ribose sugar
instead of the normal OH found in deoxynucleotide
triphosphates (dNTPs).
Therefore in a synthesis reaction, if a dideoxynucleotide is
added instead of the normal deoxynucleotide, the synthesis
stops at that point because the 3’OH necessary for the
addition of the next nucleotide is absent.
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13. DEOXY VERSUS DIDEOXY
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14. PRINCIPLE :
The sequence of a single-stranded DNA molecule
is determined by enzymatic synthesis of
complementary polynucleotide chains.
These chains terminating at specific nucleotide
positions.
Separate by gel electrophoresis
Read DNA sequence
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15. REQIREMENTS
DNA sequencing is performed in four separate tubes,
each containing
i. Single stranded DNA to be sequenced
ii. DNA polymerase
iii. Primers
iv. The four dNTPs (dATP, dCTP, dTTP and dGTP)
v. Small amount of one of the four ddNTPs
(ddATP or ddCTP or ddTTP or ddGTP)
Either the primers or the dNTPs are radiolabeled with 32P
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16. PROCEDURE
In the Sanger method, the DNA strand to be analyzed is used as a
template and DNA polymerase is used, in a PCR reaction, to
generate complimentary strands using primers.
Four different PCR reaction mixtures are prepared, each containing
a certain percentage of dideoxynucleoside triphosphate (ddNTP)
analogs to one of the four nucleotides (ATP, CTP, GTP or TTP).
Synthesis of the new DNA strand continues until one of these
analogs is incorporated, at which time the strand is prematurely
truncated.
Each PCR reaction will end up containing a mixture of different
lengths of DNA strands, all ending with the nucleotide that was
dideoxy labeled for that reaction.
Gel electrophoresis is then used to separate the strands of the four
reactions, in four separate lanes, and determine the sequence of
the original template based on what lengths of strands end with
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21. COMPARISON
Sanger Method Maxam Gilbert
Method
Enzymatic Chemical
Requires DNA synthesis Requires DNA
Termination of chain
elongation
Breaks DNA at different
nucleotides
Automation Automation is not available
Single-stranded DNA. Double-stranded or single-
stranded DNA
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Applications of DNA Sequencing
• Forensics
– Identify individuals
– Determine the paternity of a child
– Identifies endangered and protected species
• Medicine
– Detect genes that are hereditary or cause diseases
• Agriculture
– Map the genome of microorganisms

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Future of DNA Sequencing
• Projects might focus on researching:
– The links to develop lifestyle
– Genomic and cardiovascular disease
– Early detections of cancer

24. THANK YOU
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