DNA Sequencing and methods

1. DNA Sequencing
Dibya Ranjan Dalei
Adm no-9PBG/16
Dept. Of PBG, CA, BBSR, OUAT

2. SUMMARY
What is DNA sequencing
Who and when discovered
How it is prepared
Its relevant to biological
science
How long will it give benefits

3. DNA
DNA is the molecule that is the
hereditary material in all living cells.
Genes are made of DNA. A gene
consists of enough DNA to code for
one protein, and a genome is simply
the sum total of an organism's DNA.

4. What is DNA made of?
DNA is a very large molecule, made up of smaller units
called nucleotides that are strung together in a row,
making a DNA molecule thousands of times longer than it
is wide.
Each nucleotide has three parts: a sugar molecule, a
phosphate molecule, and a structure called a nitrogenous
base. The nitrogenous base is the part of the nucleotide
that carries genetic information. The bases found in DNA
come in four varieties: Adenine(A), Cytosine(C),
Guanine(G), and Thymine(T).

5. DNASequencing
DNA sequencing is the process of
determining the precise order
of nucleotides within a DNA molecule.
It includes any method or technology
that is used to determine the order of
the four bases A,T,G &C in a strand of
DNA.

6. Purpose:-

7. HISTORY:-
DNA Friedrich Miescher
James Watson Francis Crick
double-helix
Rosalind Franklin
Frederick Sanger,
sequencing of proteins the sequencing of
DNA
Frederick Sanger
insulin.
bacteriophage φX174

8. DNA sequencing methods

9. Maxam & Gilbert method
By A.M. Maxam and W.Gilbert-1977
Chemical sequencing
Treatment of DNA with certain
chemicals- DNA cuts into fragments-
monitoring of sequences

10. Procedure
1. Denature a double-stranded DNA to single-stranded by increasing
temperature.
Radioactively label one 5' end of the DNA fragment to be sequenced by a
kinase reaction using gamma-32P.
2. Cleave DNA strand at specific positions using chemical reactions. For
example, The method developed by Maxam and Gilbert uses formic acid (fire
ant venom) to break DNA after both A and G, dimethyl sulfate (toxic) to break
after G, and hydrazine (rocket fuel) to break after C and T or, if you added salt,
only after C.
3. The chemical treatments outlined in Maxam-Gilbert's paper cleaved at G,
A+G, C and C+T. A+G means that it cleaves at A, but occasionally at G as well.
4. Now in four reaction tubes, we will have several differently sized DNA
strands.
Fragments are electrophoresed in high-resolution acrylamide gels for size
separation.
5. These gels are placed under X-ray film, which then yields a series of dark
bands which show the location of radiolabeled DNA molecules. The fragments
are ordered by size and so we can deduce the sequence of the DNA molecule.

11. An example Maxam–Gilbert
sequencing reaction.
Cleaving the same tagged
segment of DNA at
different points yields
tagged fragments of
different sizes. The
fragments may then be
separated by gel
electrophoresis.

12. Sanger Method (Enzymatic)

13. Sanger method
The chain termination reaction
Dideoxynucleotide triphosphatase
(ddNTPs) chain terminators
{Having an H on the 3’C of the ribose
sugar (normally OH found in dNTPs)}
ssDNA- addition of ddNTPs-
elongation stops

14. Comparison between NTP, dNTP, &
ddNTP

15. PRINCIPLE
ssDNA (Complementary strand)
Enzymatic synthesis of complementary
polynucleotide chains
Termination at specific nucleotide positions
Separate by Gel/Capillary Electrophoresis
Read DNA sequence

18. 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

19. Automated DNA
Sequencing
Automated DNA Sequencing is based on the Sanger-Coulson method, with two
notable differences from the standard procedure. The first difference concerns the
labelling of the products of Polymerase Chain Reaction: automated produce use
fluorescent labels in the place of radioactive labelling used in the standard procedure.
The fluorescent labels are usually attached to the four dideoxynucleotides used for
chain termination. In the four track system of automated DNA Sequencing, each of the
four dideoxynucleotides used in a separate reaction, and the products are run in 4
adjacent lanes of the gel. If a different fluorochrome is attached to each of the four
dideoxynucleotides, all of them could be used in the same reaction in place of
preparing a separate reaction for each dideoxynucleotide. This is called the single track
system since the reaction products are run in a single gel lane or capillary. Generally,
the DNA to be sequenced is subjected to thermal cycle sequencing to generate the
chain terminated polynucleotides required for sequencing.

20. The reaction products are subjected to polyacrylamide gel electrophoresis under
denaturing conditions or loaded into a capillary filled with a sequencing gel. The bands
produced in the polyacrylamide gel or capillary are identified with the help of a fluorescence
detector, which identifies the fluorescent signal emitted by each band. The flurochromes are
excited by laser beam and the resulting fluorescence signal in sensed by a photovoltaic cell. The
resulting data are fed into a computer, which, in turn, converts these signals into the base
sequence of the DNA molecule. The sequence information could be printed out or stored in a
data storage device for future use; this is the second major deviation from the standard Sanger-
Coulson procedure. In the fourth track system, the sequence can be recognised from the raw
data but it has to be interpreted using an appropriate computer program in the single track
system; this becomes necessary because the shifts in mobility due to the different
fluorochromes have to be compensated for. Automated DNA sequencers can read upto 96 DNA
sequences in a 2 hours period, which is extremely fast as compared to manual DNA Sequencing.
Automated DNA Sequencing has the following advantages over manual DNA
Sequencing:(1) Radioactivity is not used,(2) Gel processing after electrophoresis and
autoradiography are not needed,(3) The tedious manual reading of gels is not required as data
are processed in a computer, (4) The sequence data is directly fed into and stored in a
computer,(5) The separation of the same reaction products can be repeated to recheck the
results in cases of doubt since they can be stored for a long period of time and (6) It is
extremely fast.

21. Applications of DNA
Sequencing
Forensics;- to help identify individuals because
each individual has a different genetic sequence.
. Medicine;- can be used to help detect the genes
which are linked to various genetic disorders such as
muscular dystrophy.
. Agriculture;- The mapping and sequencing of a
genome of microorganisms has helped to make them
useful for crops and food plants.

22. .Advantages-
.1) Improved diagnosis of disease
.2) Bio pesticides
.3) Identifying crime suspects
.4) Sequencing the whole Genome of
organisms (e.g. Human genome project)
. Disadvantages-
.1) Whole genome cannot be sequenced
at once
.2) Very slow and time consuming