The document discusses Sanger sequencing, a method of DNA sequencing. It provides a brief history of DNA sequencing, noting that Sanger developed an enzymatic DNA sequencing technique in 1977. The document then describes the key steps of Sanger sequencing, including separating the DNA strands, copying one strand with chemically altered bases that cause termination, and analyzing the fragments to reveal the DNA sequence. It also compares Sanger sequencing to the Maxam-Gilbert chemical degradation method.

1. Sanger sequencing
(Enzymatic DNA sequencing)
Jyoti Pawar
M.Sc IV sem - Biotechnology

2. Sanger sequencing Contents
• Introduction of DNA sequencing
• History of DNA sequencing
• Purpose
• Methods of DNA sequencing
Maxam & Gilbert method
Sanger method
• Advantages and Disadvantages
• Comparison
• Bibliography

3. 1. Introduction of DNA sequencing
The process of figuring out the correct order of the
four nitrogen-containing bases:
⁻ Adenine,
⁻ Guanine,
⁻ Cytosine,
⁻ Thymine in a section of DNA
“Determining the order of bases in a section of
DNA.”

4. History of DNA sequencing
“DNA sequencing method developed by Fred Sanger “
In the 1980s, two key developments allowed researchers to
believe that sequencing the entire genome could be possible.
 The first was a technique called polymerase chain reaction (PCR) that
enabled many copies of DNA sequence to be quickly and accurately
produced.
 The second, an automated method of DNA sequencing, built upon the
chemistry of PCR and the sequencing process developed by Frederick
Sanger in 1977.

5. Purpose
• Deciphering “code of life”
• Detecting mutations
• Typing microorganisms
• Identifying human halotypes
• Designating polymorphisms

6. Method of DNA sequencing
To sequence the DNA:
• Separated into two strands.
• The strand to be sequenced is copied using chemically altered
bases.
• These altered bases cause the copying process to stop each
time one particular letter is incorporated into the growing DNA
chain.
• This process is carried out for all four bases, and then the
fragments are put together like a jigsaw to reveal the sequence
of the original piece of DNA.

7. DNA sequencing Methods
Historically there are two main methods of DNA sequencing:
1. Maxam and Gilbert method
2. Sanger method
Modern sequencing equipment uses the principles of
the Sanger technique.

8. MAXAM & GILBERT METHOD
• A. M. Maxam and W.Gilbert-1977
• The sequence of a double-stranded or single-
stranded DNA molecule is determined by treatment
with chemicals that cut the molecule at specific
nucleotide positions.

9. Principle : MAXAM & GILBERT METHOD
“Chemical degradation”
Reaction in two stages:
• Chemical modification of the bases
• Modified base is removed from its sugar, pyperidin cleaves
phosphodiester bonds 5’ and 3’ and base is released.

10. SANGER METHOD
• Most common approach used to sequencing
DNA.
• Invented by Frederick Sanger - 1977
• Nobel prize - 1980
• Also termed as -
⁻ chain termination
⁻ dideoxy method
⁻ sanger sequencing

11. Principle: SANGER METHOD
“Method of DNA sequencing “
Based on the selective incorporation of chain-terminating
dideoxynucleotides by DNA polymerase during in vitro DNA
replication.
• The sequence of a single-stranded DNA molecule is determined
by enzymatic synthesis of complementary polynucleotide
chains.
• These chains terminating at specific nucleotide positions.
• Separate by gel electrophoresis
• Read DNA sequence

12. Requirements
DNA sequencing is performed in four separate tubes,
each containing
i. Single stranded DNA to be sequenced
ii. DNA polymerase
iii. Primers
iv. The four dNTPs (dATP, dCTP, dTTP and dGTP)
v. Small amount of one of the four ddNTPs (ddATP or
ddCTP or ddTTP or ddGTP)

13. Steps of Dideoxy Sequencing
1. A primer is annealed to a single-stranded section of DNA
2. DNA- primer mixture is put into 4 separate tubes with DNA
polymerase and a solution of dNTPs at a concentration of 100 times
lower than the dNTP concentration.
3. DNA Pol uses dNTPs to extend the DNA
4. ddNTPs are put together randomly, resulting in different lengths of
fragments
5. Fragments that are from each of the reactions are denatured and
separated by size using gel electrophroesis
6. The gel is used to visually detect the DNA fragments. The fragments
are to be read from bottom to top and this represents the
complementary sequence of the original strand of DNA.

14. Advantages and Disadvantages
• Allows scientists to
determine genome
sequence
• Identify the genes
causing genetic diseases
• Provides details about
individuals and their
families
• Police have DNA
samples of people that
have committed no
crime

15. COMPARISON
• Sanger Method
• Enzymatic
• Requires DNA synthesis
• Termination of chain
elongation
• Automation
• Single-stranded DNA
• Maxam Gilbert Method
• Chemical
• Requires DNA
• Breaks DNA at different
nucleotides
• Automation is not
available
• Double-stranded or
single-stranded DNA

16. Bibliography
• "Dideoxy Sequencing Animation." Dideoxy Sequencing Animation. Web. 23
May 2012.
Link <http://spine.rutgers.edu/cellbio/assets/flash/dideoxy.htm>.
• "Sanger's Method of Dna Sequencing Video." World News. Web. 23 May
2012.
Link <http://wn.com/Sanger's_Method_of_DNA_Sequencing_Video>.
• "DNA Sequencing: Method, Benefits and Applications." DNA Sequencing:
Method, Benefits and Applications. Web. 23 May 2012.
Link <http://www.biotecharticles.com/Genetics-Article/DNA-Sequencing-
Method-Benefits-and-Applications-248.html>.

17. THANK YOU