1. Sequenciamento
de DNA

2. Primer

3. 1 A 2 5’ 3’ 5’ 3’ Sanger’s Method: How Terminated OH PO4 H 5’ P R
ddNTP
Terminated
Normal Linking 3 4 5 6 A
PO4 2- H 3’ 5’ 2
Phosphodiester
bond 3’
dideoxynucleotide
Can not react

4. How DNA Sequence Is Determined?
DNA fragments having a difference
of one nucleotide can be separated
on gel electrophoresis
Polyacrylamide Gel Electrophoresis
T A G C
ATCGATCGAT
32P
ATCGATCGA
32P
ATCGATCG
32P
ATCGATC
32P
ATCGAT
32P
ATCGA
32P G
ATCG
32P C
ATC
32P T AT
32P A A
32P
But these bands can’t tell us
the identity of the terminal
nucleotides
If those band with the same
terminal nucleotide can be
grouped, then it is possible
to read the whole sequence

5. How to Obtain DNA Fragments
Maxam-Gilbert's Method:
Chemical
method
Destroy → Cleavage
32P
ATCGATCG AT
32P
ATCGATCGAT
32P
ATCG
ATCGAT
Destroy → Cleavage
Non-radioactive
(invisible)
Specific Reaction to G
Sanger's Method:
32P
Biosynthetic method
Keep on going
ATCGA
32P
A,T,C,G A
Analogue
TAGCTAGCTA
ATCG
Producing various fragments or
TAGCTAGCTA
STOP
Terminated
Template
ATCG A
TAGCTAGCTA