Gene Sequencing, a tool to analyze the exact order of nucleotide sequence in the DNA -Deoxyribonucleic Acid. Focuses on Two methods: a. Maxam-Gilbert (Chemical Degradation) Method b. Sanger's Method (Dideoxy Chain termination Method)

1. GENE SEQUENCING
PREPARED BY:
Sajin Dangol
MSc. Microbiology
St. Xavier’s College, Kathmandu

2. INTRODUCTION
• DNA is the blueprint of life consisting of chemical building
blocks called nucleotides.
• These building blocks are made of three parts:
phosphate, sugar group, and one of the four types of
nitrogen bases viz adenine (a), thymine (t), guanine (g), or
cytosine (c).
• The order or sequence of these bases determines
what biological instructions are contained in a strand
of dna.

3. • DNA is a double helix
• Two strands run anti parallel to each other
• Paired together by hydrogen bonds
• Highly specific nature of this type of chemical pairing
• A always pairs with base T, and likewise C with G
• Therefore, if the sequence of the bases on one strand of a
DNA double helix is known, it is simple to figure out the
sequence of bases on the other strand.

4. Landmarks in DNA Sequencing
• 1953 Discovery of the structure of the DNA double
helix.
• 1972 Development of recombinant DNA technology.
• 1977 The first complete genome of bacteriophage uX174
sequenced.
• 1977 Allan Maxam and Walter Gilbert publish ‘‘DNA
sequencing by chemical degradation.
• 2001 A draft sequence of the human genome published.

6. What is Gene Sequencing
• DNA sequencing is the process of determining the exact
order of nucleotides within a DNA molecule. This method is
used to determine the order of the four bases—adenine (A),
guanine (G), cytosine (CY), and thymine (T) in a strand of
DNA.

8. Maxam–Gilbert Method
• Allan Maxam and Walter Gilbert developed a method
for sequencing single-stranded DNA by a two-step
catalytic process involving piperidine and two
chemicals that selectively attack purines and
pyrimidines .

9. • This is the best method offered for sequencing of small
oligonucleotides.
• Maxam–Gilbert sequencing is a method of DNA
sequencing
developed by Allan Maxam and Walter Gilbert in 1976.
• Maxam–Gilbert sequencing requires radioactive labeling
at
one 5′ end of the DNA fragment to be sequenced.
• The Maxam and Gilbert chemical sequencing method is
based on the ability of different chemical to specifically
modify bases within the DNA molecule.
• The chemical specificity of the first reactions are as
follows:
G: DMS
G+A: Formic acid
T+C: Hydrazine
C: In the presence of NaCl, only C reacts with hydrazine

10. Procedure (STEPS)
• Radioactive labelling of one end (5' end or 3’ end) of the
DNA fragment to be sequenced by a kinase reaction using
32P.
• Cut the DNA fragment with specific restriction enzyme,
resulting in two unequal DNA fragments
• Denature the double-stranded DNA to single-stranded
DNA by increasing temperature.
• Cleave the DNA strand at specific positions using
chemical reactions.

11. We can use one of the two chemicals followed by
addition of piperdine. Dimethyl sulphate (DMS)
selectively attacks purine (A and G), while hydrazine
selectively attacks pyrimidines (C and T). This is called
modification step.
• Chemical treatment generates breaks at the four
nucleotide bases in the four reaction mixtures (G,
A+G, C, and C+ T).

13. • Fragments are subjected to electrophoresis in high-
resolution acrylamide gels for size-based separation.
• To visualize the fragments, the gel is exposed to X-
ray film for autoradiography, which yields a series of
dark bands, each corresponding to a radiolabeled
DNA fragment, from which the nucleotide sequence
may be inferred.
• In the gel, the fragments are ordered by size and,
thus, we can deduce the sequence of the DNA
molecule.

15. Sanger and Coulson Method
Dideoxynucleotide Chain
Termination Method
• It is an enzymatic method.
• developed by Frederick Sanger and his colleagues in
1977.
• Sanger won the Nobel Prize in Chemistry in 1980 for
development of this technique.
• A powerful technique for sequencing.
• Utilizes single stranded DNA as a template.

16. • Also called dideoxynucleotide termination method.
• Requirements:
• A primer with free 3’-OH ends to start DNA
synthesis
• DNA Polymerase
• dNTPs

19. Feature of ddNTPs
• If any of the ddNTPs binds, the chain elongation is
terminated.
• Because ddNTPs don’t have free 3’-OH end which is
required for chain elongation.
• Therefore, no phosphodiester bond will be formed.

21. Procedure
• Four reaction tubes are labelled with A,T,G and C
each containing -
• Single stranded DNA template – obtained by NaOH
hydrolysis
• 5’-radiolabelled DNA primer and,
• All four radiolabelled dNTPs (dATP, dGTP, dCTP
and dTTP)

23. • A small amount of:
• ddATP is added to tube 1,
• ddGTP to tube 2,
• ddCTP to tube 3 and,
• ddTTP to tube 4.
• Conentration of ddNTPs should be maintained to
about 1% of the concentration of dNTPS

24. Procedure Contd…
• DNA polymerase is added to each tube.
• DNA synthesis starts and chain elongates.
• In each tube ddNTP is randomly incorporated and
fragments are terminated.
• The length of fragments depend on the position of
incorporation of ddNTPs.

25. • After completion of reaction, the fragments of each
tubes are separated by electrophoresis in four
different lanes of high resolution Polyacrylamide
Gel.
• Gel is then dried and autoradiography is done.
• DNA sequence is obtained by reading the bands on
autoradiogram of four lanes.
• (from bottom to top of gel)

28. Significance of DNA Sequencing
• Information obtained by DNA sequencing makes it
possible to understand or alter the function of genes.
• DNA sequence analysis demonstrates regulatory
regions that control gene expression and genetic “hot
spots” particularly susceptible to mutation.
• Comparison of DNA sequences shows evolutionary
relationships that provide a framework for definite
classification of microorganisms including viruses.

29. • Comparison of DNA sequences facilitates identification
of conserved regions, which are useful for development
of specific hybridization probes to detect microorganisms
including viruses in clinical samples.
• DNA sequencing has become sufficiently fast and
inexpensive to allow laboratory determination of
microbial sequences for identification of microbes.
Sequencing of the 16S ribosomal subunit can be used to
identify specific bacteria. Sequencing of viruses can be
used to identify the virus and distinguish different strains.
• DNA sequencing shows gene structure that helps research
workers to find out the structure of gene products.