1. 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 ATC GATC GAT
32
P ATC GATC GA
32
P ATC GATC G
32
P ATC GATC
32
P ATC GAT
32
P ATC GA G
32
P ATC G 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

2. How to Obtain DNA Fragments
Maxam-Gilbert's Method: Chemical Destroy → Cleavage
method
32
P ATC GATC G AT
32
P ATC GATC GAT
32
P ATC G ATC GAT
Non-radioactive
Specific Reaction to G (invisible)
Destroy → Cleavage
Sanger's Method: Biosynthetic method 32P Keep on going
32
P Analogue ATC GA
A,T,C ,G A TAGC TAGC TA
ATC G or Producing various fragments
TAGC TAGC TA STOP Terminated
Template ATC G
A
TAGC TAGC TA
Juang RH (2004) BCbasics

3. 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
PO 4 A R 4
P
2
Phosphodiester
bond R 5
P
3’ H H R 6
dideoxynuceotide Can not react 3’
Juang RH (2004) BCbasics