ppt on nucleotide sequencing

1. SUBMITTED TO,
Dr. Monika Asthana
Incharge
Department Of Biotechnology
SUBMITTED BY,
Prashant sharma
M.Sc. Biotechnology
2nd Semester

2. WHAT IS 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.
The DNA has four bases i.e;
• Adenine (A)
• Thymine (T)
• Cytosine (C)
• Guanine (G)

3. WHAT IS DNA SEQUENCING
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

4. HISTORY
• The sequencing of DNA
molecules began in the 1970s
with development of the
MaxamGilbert ethod, and
later the Sanger method.
• Originally developed by
Frederick Sanger in 1975.
• 1987 – Applied biosystems
marketed Fully automated
sequencing machines

5. METHOD’S OF DNA SEQUENCING
• Historically , there are two main methods i.e;
1- Maxam Gilbert method or Chemical cleavage method
2- Sangers method or chain termination method
• Some other sequencing methods are –
1- Automated sequencing
2- Whole genome sequencing
3- Next generation sequencing

6. MAXAM GILBERT SEQUENCING
METHOD
• By A.M. Maxam and W.Gilbert-1977
• Chemical sequencing Treatment of DNA with certain
chemicals
• DNA cuts into fragmentsmonitoring of sequences

7. PRINCIPLE
• The partially cleaved DNA fragment is subjected to
five separate chemical reactions.
• Each of which is specific for a particular base.
• The resulting fragment terminate at that specific
base followed by high resolution gel electrophoresis
and detection of the labeled fragments by
autoradiography

8. METHOD
• Labeled DNA at one
end with 32P.
• DNA copies are
divided in to 4
samples.
• Each samples treated
with a chemical that
specifically destroys
one or two of the 4
base in DNA.

9. PROCEDURE

10. SANGER’S METHOD
• Most common approch
used for DNA
sequencing.
• Invented by “Frederick
Sanger” in 1977.
• Noble prize in -1980.
• Also termed as Chain
termination method or
Dideoxy method.

11. PRINCIPLE
• Enzymatic synthesis of complementary
polynucleotide chains
• Termination at specific nucleotide positions
• Separate by Gel/Capillary Electrophoresis
• Read DNA sequence

12. REQUIREMENTS
• Single Stranded template
• Primer
• DNA polymerase
• Di-Deoxynucleotide
• The 3′-OH group necessary for formation of the
phosphodiester bond is missing in ddNTPs)
• Every nucleotide have its specific ddNTP form i.e.,
ddATP, ddGTP etc

13. METHOD
Steps:
1. Denaturation
2. Primer attachment
and extension of bases
3. Termination
4. Gel electrophoresis

14. PROCEDURE

15. AUTOMATED SEQUENCING METHOD
• Florescence technique is used for detection of DNA
bands.
• Fluorescent labbeled dd NTP are used.
• Resulting DNA strands are seperated in 4 different
lane in electrophoresis.
• Detected by fluorescence detector.

17. WHOLE GENOME SEQUENCING METHOD
SHORTGUN SEQUENCING METHOD
Shotgun sequencing, also known as shotgun cloning, is
a method used for sequencing long DNA strands or the
whole genome.

18. NEXT GENERATION SEQUENCING
METHODS
• The next generation sequencing is based upon the
principle of Sanger’s method .
• There are many types of NGS are present I.e;
1- Pyrosequencing
2- Illumina sequencing
3- Solid sequencing etc.

19. ADVANTAGES / DISADVANTAGES
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

20. APPLICATIONS
• 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.