SANGER'S METHOD: DNA sequncing and detection by the gel electrophrosis

1. NAME: NAYA SAEED-057
TOPIC: METHODS OF SANGER’S METHOD

2. Sanger’s Method
PRINCIPLE
 The sequence of a single stranded DNA molecule is
determined by enzymatic synthesis of complementary
polynucleotide chains
 These chains terminating at specific nucleotide position
 Separate by Gel electrophoresis
 Read DNA sequence

3. REQUIREMENTS
DNA sequencing is performed in four separate
tubes each containing.
1) Single strandedDNA to be sequenced
2) DNA polymerase
3) The four dNTPs (dATP, dCTP, dTTP and dGTP)
4) Small amount of one of four ddNTPs (ddATP
or ddCTP or ddTTP or ddGTP)

4. Principle

6. METHODOLOGY
• It depends on the fact that:
A) Synthesis of a double-stranded DNA
segment from a single strand of DNA will be
initiated in the presence of DNA polymerase,
B) DNA synthesis will stop if the incorporated
base is in the form dideoxynucleotide instead of
deoxynucleotide.

7. CONTINUE
• Elongation of strand Random addition of
dNTPs and ddNTPs.
• When ddNTP added strand extension
terminates
• Being random - all possible lengths of the
DNA fragments are generated having a
terminal fluorescence labeled ddNTP.
• Product of this reaction is subjected to gel
electrophoresis

8. Sanger’s Method