9/25/2013 1MALLIGE COLLEGE OF PHARMACY
Deoxyribonucleic acid (DNA) is a nucleic acid that
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
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),
9/25/2013 2MALLIGE COLLEGE OF PHARMACY
Watson & Crick Model
9/25/2013 3MALLIGE COLLEGE OF PHARMACY
Determining the order of bases in a section
9/25/2013 4MALLIGE COLLEGE OF PHARMACY
• Deciphering “code of life”
• Detecting mutations
• Typing microorganisms
• Identifying human halotypes
• Designating polymorphisms
9/25/2013 5MALLIGE COLLEGE OF PHARMACY
DNA SEQUENCING METHODS
Historically there are two main methods of
1.Maxam and Gilbert method
Modern sequencing equipment uses the
principles of the Sanger technique.
9/25/2013 6MALLIGE COLLEGE OF PHARMACY
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.
9/25/2013 7MALLIGE COLLEGE OF PHARMACY
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
9/25/2013 8MALLIGE COLLEGE OF PHARMACY
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.
9/25/2013 9MALLIGE COLLEGE OF PHARMACY
• Most common approach used to
• Invented by Frederick Sanger - 1977
• Nobel prize - 1980
• Also termed as chain termination or
9/25/2013 11MALLIGE COLLEGE OF PHARMACY
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
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.
9/25/2013 12MALLIGE COLLEGE OF PHARMACY
DEOXY VERSUS DIDEOXY
9/25/2013 13MALLIGE COLLEGE OF PHARMACY
The sequence of a single-stranded DNA molecule
is determined by enzymatic synthesis of
complementary polynucleotide chains.
These chains terminating at specific nucleotide
Separate by gel electrophoresis
Read DNA sequence
9/25/2013 14MALLIGE COLLEGE OF PHARMACY
DNA sequencing is performed in four separate tubes,
i. Single stranded DNA to be sequenced
ii. DNA polymerase
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
9/25/2013 15MALLIGE COLLEGE OF PHARMACY
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
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
what nucleotide.9/25/2013 16MALLIGE COLLEGE OF PHARMACY
Sanger Method Maxam Gilbert
Requires DNA synthesis Requires DNA
Termination of chain
Breaks DNA at different
Automation Automation is not available
Single-stranded DNA. Double-stranded or single-
9/25/2013 21MALLIGE COLLEGE OF PHARMACY
9/25/2013 MALLIGE COLLEGE OF PHARMACY 22
Applications of DNA Sequencing
– Identify individuals
– Determine the paternity of a child
– Identifies endangered and protected species
– Detect genes that are hereditary or cause diseases
– Map the genome of microorganisms
9/25/2013 MALLIGE COLLEGE OF PHARMACY 23
Future of DNA Sequencing
• Projects might focus on researching:
– The links to develop lifestyle
– Genomic and cardiovascular disease
– Early detections of cancer
9/25/2013 24MALLIGE COLLEGE OF PHARMACY