Detailed explanation about gene sequencing methods Sequencing the gene is an important step toward understanding the gene. A gene sequence contains some clues about where genes are. Gene sequencing give us understanding how the genome as a whole works-how genes work together to direct the growth, development and maintenance of an entire organism. It help scientists to study the part of genome outside the genes-regulatory regions

1. GENE SEQUENCING
BY BAIDEHI MITRA
MPHARM (PHARMACOLOGY)

2. WHAT IS GENE SEQUENCING?
 Sequencing means to determine the primary structure of an
unbranched biopolymer.
 Gene sequencing is the technique that allows researchers to read
the genetic information found in DNA. Sequencing involves
determining the order of bases.

3. IMPORTANCE OF GENE SEQUENCING
 Sequencing the gene is an important step toward
understanding the gene.
 A gene sequence contains some clues about where genes
are.
 Gene sequencing give us understanding how the
genome as a whole works-how genes work together to
direct the growth, development and maintenance of an
entire organism.
 It help scientists to study the part of genome outside the
genes-regulatory regions

4. TYPES OF GENE SEQUENCING:
Automated
method
Sangers
sequencing
method
pyrosequencin
g
Maxam gilbert method

5. SANGER SEQUENCING INTRODUCTION
 Sanger method used in the In-vitro
DNA replication.
 Sanger sequencing technique is
developed by British biochemist
Frederick and his team in 1977 .
 Frederick Sanger received Nobel prize
for development of Sanger sequencing
1980.

6. PRINCIPLE
 Sanger sequencing is based on
di deoxy nucleotides in DNA
polymerizing reaction.
 The nucleotide has three
components, the nitrogenous
base, which can either be A T G or
C, the sugar residue and the
phosphate attached with the
sugar residue. When all three
components are present, it's
called nucleotide.
 If the phosphate is absent, then
it's called nucleoside.

7.  If the hydroxyl group at the 2’
end of the sugars removed, then
it's called deoxy nucleotide
 Deoxy Nucleotide are present in
DNA. Substrate for various DNA
polymerase.
 The nucleotide having 3-OH
group absent is called di deoxy
nucleotides.
Deoxyribose sugar-------------Binds to
nucleotide
Di deoxyribose sugar----------------
cannot bind to the nucleotide.
Therefore, if 3’OH GROUP IS ABSENT THERE
WON’T BE NEW NUCLEOTIDE IN THE CHAIN.

8. Agarose

9. dATP (adenine)
dTTP (Thymine)
dGTP (Guanine)
dCTP (Cytosine)
ddATP
ddTTP
ddGTP
ddCTP
dideoxyribonucleotid
e
deoxyribonucleotide

10. • Take 4 test tubes because we have 4 nucleotides. The DNA sample to be sequenced is
combined in 4 tube with primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and
dCTP) and ddNTPs
• The mixture is first heated to denature the template DNA at 95℃ (separate the strands), then
cooled so that the primer can bind to the single-stranded template.
To produce a
complementary
strand and the
template strand for
DNA sequencing.

11. • A primer is then annealed to the 3’ end of the DNA
5’
3’
Note: Primer will run from 5’ to 3’ prime
DNA POLYMERASE ADDED, THEN THIS WILL BE
RUNNING FROM 3’ PRIME TO 5’ PRIME DIRECTION
OF THE TEMPLATE STRAND

12. • As it moves towards the forward direction then it starts adding the
nucleotides.
• DNA polymerase/ primer accepts these nucleotides (dNTPs /ddNTPs).
• This will extend the primer. This process is called
extension.
• After all the acceptance of nucleotides.

13. • So first test tube----- only 3 primers produced.
• The same process we will do with 2, 3 and 4 test-tube, only change
in ddNTPs.

14. • As a result of the chain
termination, DNA
fragments of different
lengths are formed
across all the reaction
vessels.
• After the reaction is done, agarose
gel electrophoresis is used to
sequence the DNA.

15. • Short fragments move quickly through
the pores of the gel, while long
fragments move more slowly.
• The smaller, lighter lengths of
DNA migrate further to the
bottom of the electrophoresis
plate.
• DNA migrates from the negative pole
towards the positive pole, due to the
negative charge imparted by their
phosphate backbone.
This is why we
see these band
patterns along
the lengths of
the plate.

16. • The sequence is read from the bottom of
the plate.
A G C T G A T A G G C A C T T A T G G T A C T
This whole process results in the
complementary sequence of the DNA sample.

18. ADVANTAGES:
o Long read lengths: up to – 700 bp.
o Easy to analyze
o Relatively small data storage required.
o Most flexible: from 1 to 1000 samples.
DISADVANTAGES:
o Expensive
o Requires PCR.
o Low sensitivity.
o Low detection rate of alternative allele.

19. MAXAM GILBERT SEQUENCING
THE CHEMICAL DEGRADATION, IN WHICH SEQUENCE OF A DNA MOLECULE IS
DETERMINED BY TREATMENT WITH CHEMICALS THAT CUT THE MOLECULE AT
SPECIFIC NUCLEOTIDE POSITIONS.
Maxam Gilbert sequencing
technique developed by
Allan Maxam and Walter
Gilbert in 1976.

20. MAXAM-GILBERT DNA SEQUENCING METHOD:
1.END LABELING
2. RESTRICTION ENZYME
3. DENATURATION
4. CHEMICAL DEGRADATION
5. GEL ELECTOPHORESIS
6. AUTORADIOGRAPHY
7. SEQUENCE DETERMINATION

21. End labeling: The labeling of DNA molecule by radioactive isotope at
the both end of the molecule.
ALKALINE
PHOSPHATASE
dephosphorylation
OH
ATP
ADP
P
Polynucleotide
kinase
Cut with Restriction
enzyme
Sequence
d
Discarded

22.  DENATURATION: BY APPLYLING HEAT AT 95℃ .
 We get single stranded form. Isolate this single stranded DNA that contain the 5’ prime
END LABELED ssDNA
CHEMICAL DEGRADATION:
3’OH
REACTION MIXTURE
TUBE

23. DIMETHYL
SULAFTE IN
FORMIC
ACID
DIMETHYL
SULAFTEWI
TH HEAT
HYDRAZINE HYDRAZINE
IN 2ML Nacl
PIPERIDINE PIPERIDINE PIPERIDINE PIPERIDINE
We obtain different fragments,
depending on the combination of
chemicals.

24. THESE REACTIONS ARE LOADED ON GEL ELECTROPHORESIS.
1. THE NEGATIVE CHARGE OF ITS PHOSPHATE BACKBONE MOVES THE DNA
TOWARDS THE POSITIVELY CHARGED ANODE.
2. SMALLER DNA MOECULES MIGRATE MORE RAPIDLY THAN LARGER
FRAGMENTS
THE DNA BAND
VISUALIZED BY
AUTORADIOGRAHY
USING X-RAY FILM
• SO WE CAN SEE THE
BANDS.
• TO DETERMINE THE
SEQUENCE WHICH ARE
PRESENT IN DNA.

25. FRAGMENT SEQUENCES ARE IDENTIFIED
• LIMITATIONS:
• WE USE RADIOACTIVE/ TOXIC CHEMICAL
• WE CAN ONLY SEQUENCE UPTO 500 BASE PAIRS

27. ADVANTAGES:
• Purified DNA can be read directly
• Can be used to analyze DNA protein interactions (i.e. footprinting)
• Can be used to analyze nucleic acid structure.
DISADVANTAGES:
• It requires extensive use of hazardous chemicals.
• It has a relatively complex set up / technical complexity.
• It is difficult to “scale up” and cannot be used to analyze more than 500 base
pairs.
• The read length decreases from incomplete cleavage reactions.
• It is difficult to make Maxam-Gilbert sequencing based DNA kits.

28. NEW GENERATIONS:
AUTOMATED DNA SEQUENCING
 The manual sequencing technique is slow and ineffective for sequencing
large genomes.
 To sequence large amount of DNA, automated sequencing method is
required.
 Automated DNA sequencing is based on original sanger method i.e. chain
termination method with a slight modification.
 It uses dideoxy nucleotides, like sanger method, with one exception.
 Di-deoxy nucleotides are tagged with different fluorescent molecules, so the
products from each tube will emit a different color fluorescence when excited
by light.

29. REQUIREMENTS:
• Template DNA i.e. DNA to be sequenced.
• DNA nucleotides dNTPs; dATP, dTTP, dCTP, dGTP.
• Primer
• DNA polymerase.
• di-deoxy nucleotides or chain terminators (ddNTPs) with a specific fluorescent
molecule.
• After carrying out DNA synthesis in four tubes all reaction products are mixed and
electrophoresed together in the same lane on a gel--Capillary gel electrophoresis.
• Fragments are separated according to their sizes.
• A laser beam illuminate the fluorescent ddNTPs and a detector identify the color.
• The information then passes to a computer which has been programmed to
convert the color information to a base sequence.
• For example, blue may indicate di-deoxy C, Green may indicate A. red indicate T
and yellow indicate G.

31. PYROSEQUENCING
 Pyrosequencing is the method of gene sequencing based on the “Sequencing by synthesis”
principle. The technique was developed by Mostafa Ronaghi and Pal Nyren at the Royal
Institute of Technology in Stockholm in 1996. It differs from Sanger sequencing, in that it
relies on the detection of pyrophosphate release on nucleotide incorporation rather than the
chain termination with dideoxynucleotides.
 Pyrosequencing is the important type of is the method of gene sequencing methodology that
is in use today. It does not require electrophoresis or any other fragment separation
procedure and so more rapid.
 Can read up to 300-500 base pairs.
 Involves the detection of visible light at 560 nm.
 Has high sensitivity.

32. PYROSEQUENCING METHOD:

35. ADVANTAGES OF PYROSEQUENCING:
o A rapid method for the analysis of individual base polymorphisms---
- 4500 can be analyzed.
o Accurate.
o Very fast.
o Low cost per base.
o Easily automated
o No need for gel electrophoresis.
o The reagents cost are lower for sequencing short stretches of DNA
compared to current available methods.
DISADVANTAGES OF PYROSEQUENCING:
o Expensive
o This is not a procedure for simple research laboratories.
o Doesn’t work well for de novo sequencing of large genomes.
o Difficulty sequencing homopolymers accurately.
o Relatively high error rate.

36. NEW GENERATION SEQUENCING METHODS
Sequencing by Ligation(SBL)
Nano pore DNA Sequencing
Ion Torrent semiconductor Sequencing
Illumina(Solexa ) Sequencing
Transmission Electron Microscopy for DNA
Sequencing
Difference

38. THANK YOU