Dna Sequencing

1. PRESENTED BY
MARIAM RAZI
BS medical technology 6
th
semester
DNA
SEQUENCING

2. DNA
•Deoxyribonucleic acid (DNA) is a nucleic
acid that functions include
Storage of genetic information
Self-duplication & inheritance.
Expression of the genetic message.
•DNA’s major function is to code for
proteins.
•Information is encoded in the order of the
nitrogenous bases.

3. Watson & Crick Model
•DNA is composed of 2 chains of nucleotides that form a
double helix shape.
•The two strands are antiparallel.
•The backbone of the DNA molecule is composed of
alternating phosphate groups and sugars.
•The complimentary nitrogenous bases form hydrogen
bonds between the strands.
•A is complimentary to T and G is complimentary to C.

4. DNA SEQUENCING
Determining the order of bases in a section
of DNA

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

6. 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.

7. 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.

8. PRINCIPLE :
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

11. SANGER METHOD
•Most common approach used to
sequencing DNA.
•Invented by Frederick Sanger - 1977

•Nobel prize - 1980
•Also termed as chain termination or
dideoxy method

12. SANGER METHOD TERMED AS
CHAIN TERMINATION METHOD
This method uses dideoxynucleotide triphosphates
(ddNTPs) chain terminators :
which have an H on the 3’ carbon of the ribose sugar
instead of the normal OH found in deoxynucleotide
triphosphates (dNTPs).

Therefore in a synthesis reaction, if a dideoxynucleotide is
added instead of the normal deoxynucleotide, the synthesis
stops at that point because the 3’OH necessary for the
addition of the next nucleotide is absent.

13. DEOXY VERSUS DIDEOXY

14. PRINCIPLE :
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

15. REQIREMENTS
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)
Either the primers or the dNTPs are radiolabeled with 32P

16. HOW DOES THE DNA TEMPLATE
OBTAINED?
•The DNA can be cloned in a plasmid vector.
•The DNA can be cloned in a bacteriophage M13
vector.
•PCR can be used to generate single-stranded
DNA.

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

19. Next Generation Technologies:
Solexa : Based whole genome sequencing
SOLiD : Ligation and detection
454 : Pyrosequencing
Helicos : Single molecule sequencing

20. THANK YOU