How DNA Sequence Is Determined?

DNA fragments having a difference

of one nucleotide can be separated Polyacrylamide Gel Electrophoresis
on gel electrophoresis T A G C
32
P ATCGATCGAT
32
P ATCGATCGA
32
P ATCGATCG
32
P ATCGATC
32
P ATCGAT
32
P ATCGA G
32
P ATCG C
32
P ATC
T
32
P AT
32
P A A
But these bands can’t tell us If those band with the same
the identity of the terminal terminal nucleotide can be
nucleotides grouped, then it is possible
to read the whole sequence
Juang RH (2004) BCbasics
 How to Obtain DNA Fragments
Maxam-Gilbert's Method: Chemical Destroy → Cleavage
method
32
P ATCGATCG AT
32
P ATCGATCGAT
32
P ATCG ATCGAT
Non-radioactive
Specific Reaction to G (invisible)
Destroy → Cleavage

Sanger's Method: Biosynthetic method 32P Keep on going

32
P Analogue ATCGA
A,T,C,G A TAGCTAGCTA
ATCG or Producing various fragments
TAGCTAGCTA STOP Terminated

Template ATCG
A
TAGCTAGCTA
Juang RH (2004) BCbasics
Sanger’s Method: How Terminated
5’
P
R 1
5’
1 P A
ddNTP R 2 Terminated
3’
Normal Linking

OH P
3
2-
5’ R
P
PO4 A R 4
P
2
Phosphodiester
bond R 5

P
3’ H H R 6

dideoxynuceotide Can not react 3’

Juang RH (2004) BCbasics