The document details the steps and methodologies for DNA sequencing, emphasizing its significance for genome mapping and manipulation in recombinant DNA experiments. It outlines traditional methods such as Sanger and Maxam-Gilbert sequencing, and introduces next-generation sequencing (NGS) as a transformative technology that allows for efficient and large-scale sequencing. Important mechanisms, including chain termination and sequencing by synthesis, are explained in the context of their application to modern genomic studies.

1. DNA sequencing

2. Steps to map the GEnome
Markers to
generate
highly
saturated map
Physical map
Sequencing
for the whole
genome

3. Steps to map the GEnome
Markers to
generate
highly
saturated map
Physical map
Sequencing
for the whole
genome

4. Why Genome Sequencing?

5. For many recombinant DNA experiments,
knowledge of a DNA sequence is a prerequisite for
its further manipulation
1. DNA sequencing followed by computer-assisted searching for restriction
endonuclease cleavage sites is often the fastest method for obtaining a
detailed restriction map.
2. Computer-assisted identification of protein-coding regions (ORF) within
the DNA sequence followed by computer-assisted similarity searches of
DNA and protein databases can lead to important insights into the
function and structure of a cloned gene and its product.
3. The DNA sequence is a prerequisite for a detailed analysis of the 5’ and 3’
noncoding regulatory regions of a gene.
4. DNA sequence information is essential for site-directed mutagenesis.
5. Small amounts of DNA sequence information ( “STS” or “EST “) are the
basis of methods for mapping and ordering large DNA segments cloned
into YACs, BACs, or cosmids

6. Main methods for sequencing DNA
The Enzymatic method
Dideoxy or Chain –
termination method
(developed by Sanger and
Coulson)
The Chemical method
(developed by Maxam and
Gilbert)
First Generation sequencing
= Maxam-Gilbert sequencing
=Sanger sequencing
=chain termination sequencing

7. THE DIDEOXY or
ENZYMATIC SEQUENCING METHOD
• based on DNA synthesis in the presence of dideoxynucleotides.
• a DNA polymerase is utilized to synthesize
➢a labeled
➢complementary copy of the DNA template
• based on electrophoretic procedures using high – resolution
denaturing polyacrylamide gels (sequencing gels)
• electropherograms

8. THE DIDEOXY or
ENZYMATIC SEQUENCING METHOD

10. Single-stranded DNA template for the sequence of
interest
DNA polymerase
a section of labeled primer
the 4 normal deoxynucleotides triphosphate (dNTPs )
ddATP ddTTP ddCTP ddGTP

12. Manual

13. Single-stranded DNA template for the sequence of
interest
DNA polymerase
a section of labeled primer
the 4 normal deoxynucleotides triphosphate (dNTPs )
ddATP
ddTTP
ddCTP
ddGTP
Each with
different
fluorescent
label

16. chain termination sequencing
methodology
standard chain termination
• employs radioactive labels
• visualized by autoradiography
• Four reaction tubes
Automated sequencing
• Employs fluoro-labeling
• visualized by fluorescent
detector which can discriminate
between the different labels
• Safer than using isotopes
• Single tube

17. THE CHEMICAL SEQUENCING METHOD
• only end-labeled fragments are observed following
autoradiography of the sequencing gel
• based on the ability of Hydrazine, Dimethyl sulfate
(DMS) or Formic acid to specifically modify bases
within the DNA molecule

18. THE CHEMICAL SEQUENCING METHOD
DMS (G) methylates nitrogen 7 (N7) of G, which then
opens between carbon 8 and nitrogen 9
(Piperidine then displaces the modified G
from its sugar)
Formic acid (G+A) weakens A and G glycosidic bonds
(The purines can then be displaced with
piperidine)
Hydrazine (T+C) splits the rings of T and C.
(The fragments then displaced by piperidine)
NaCl (C) In the presence of NaCl, only C reacts with
hydrazine
(The modified C can then be displaced with
piperidin)
Piperidine catalyze strand breakage at these modified
nucleotides.

20. What is the difference
between generated
sequence from sanger
and Maxam-Gilbert?

21. Next-Generation Sequencing (NGS)
• Despite many technical improvements during this era, the limitations
of automated Sanger sequencing showed a need for new and
improved technologies for sequencing large numbers of human
genomes
• The major advance offered by NGS is the ability to produce an
enormous volume of data cheaply - in some cases over one billion
short reads per instrument run

22. Sequence By Synthesis(SBS)
Pal Nyren's Pyrosequencing Method
1. a non-electrophoretic
2. real-time DNA-sequencing method
• Based on sequencing by synthesis that relies on the detection of
Pyrophosphate (PPi) released during the DNA polymerization reaction
• Pyrosequencing® technology is sequencing by synthesis, a simple-to-
use technique for accurate and quantitative analysis of DNA
sequences.

23. Sequence By Synthesis(SBS)
Pal Nyren's Pyrosequencing Method
4 Enzymes 2 substrates
Apyrase
luciferase
ATP
sulfurylase
DNA
polymerase
adenosine 5”
phosphosulfate
(APS)
luciferin

24. Step 1
Step 2
Step 3
Step 4
Step 5
A sequencing primer is hybridized into a single
stranded PCR amplicon that serves as a template,
and incubated with the enzymes:
1. DNA polymerase
2. ATP sulfurylase
3. Luciferase
4. Apyrase
substrates:
1. (APS)
2. luciferin.

25. Step 1
Step 2
Step 3
Step 4
Step 5
The first deoxyribonucleotide triphosphate (dNTP)
is added to the reaction .
■ DNA polymerase catalyzes the incorporation of
the deoxyribonucleotide triphosphate into the
DNA strad, if it is complementary to the base in
the template strand.
■ Each incorporation event is accompanied by
release of pyrophosphate (PPi) in a quantity
equimolar to the amount of incorporated
nucleotide.

26. Step 1
Step 2
Step 3
Step 4
Step 5
ATP sulfurylase converts PPi to ATP in the presence of
adenosine 5' phosphosulfate (APS).
■This ATP drives the luciferase-mediated conversion of
luciferin to oxyluciferin that generates visible light in
amounts that are proportional to the amount of ATP.
■The light produced in the luciferase-catalyzed
reaction is detected by a charge-coupled device (CCD)
chip and seen as a peak in the raw data output
(Pyrogram).
■The height of each peak (light signal) is proportional
to the
number of nucleotides incorporated.

27. Step 1
Step 2
Step 3
Step 4
Step 5
Apyrase, a nucleotide-degrading enzyme, continuously
degrades unincorporated
nucleotides and ATP.
• When degradation is complete, another nucleotide is
added.

28. Step 1
Step 2
Step 3
Step 4
Step 5