Biomedical Engineering Reference
In-Depth Information
Base
(A, G, C or T)
Base
(A, G, C or T)
O
O
O
O
O
O
5'
5'
HO
P
O
P
O
P
O
CH
2
HO
P
O
P
O
P
O
CH
2
O
O
O
-
O
-
O
-
O
-
O
-
O
-
4'
1'
H
4'
1'
H
H
H
H
H
H
H
3'
2'
3'
2'
OH
H
OH group missing
H
H
Normal deoxynucleoside triphosphate
(i.e. 2' deoxynucleotide)
Dideoxynucleoside triphosphate
(i.e. 2', 3' dideoxynucleotide)
Fig. 7.1
Dideoxynucleoside triphosphates act as chain terminators because they lack a 3
′
OH group. Numbering of the carbon
atoms of the pentose is shown (primes distinguish these from atoms in the bases). The
α
,
β
and
γ
phosphorus atoms are indicated.
5'
3'
CATA
CG
TGG CCTTACG
Add 5'CGT
AAG
G3
'
primer
5'
3'
CATACGT
GG
CCTTACG
GGAATGC 5'
Add d*ATP, d*CTP, d*GTP and d*TTP
Add
ddATP
+
Klenow
fragment
Add
ddCTP
+
Klenow
fragment
Add
ddGTP
+
Klenow
fragment
Add
ddTTP
+
Klenow
fragment
CGTAAGG*C*C
dA
and
CGTAAGG*C*C*A*G*G*T
dA
CGTAAGG*C*C*A*C
dG
and
CGTAAGG*C*C*A*C*G*T*A*T
dG
CGTAAGG
dC
and
CGTAAGG*C
dC
and
CGTAAGG*C*C*A
dC
CGTAAGG*C*C*A*C*
GdT
and
CGTAAGG*C*C*A*C*G*T*
AdT
Electrophorese and autoradiograph
Sequence
CGTAAGGCCACGTAT
dG
CGTAAGGCCACGTA
dT
CGTAAGGCCACGT
dA
CGTAAGGCCACG
dT
CGTAAGGCCAC
dG
CGTAAGGCCA
dC
CGTAAGGCC
dA
CGTAAGGC
dC
CGTAAGG
dC
ACGT
Fig. 7.2
DNA sequencing with
dideoxynucleoside triphosphates as
chain terminators. In this figure
asterisks indicate the presence of
32
P
and the prefix 'd' indicates the presence
of a dideoxynucleoside. At the top of
the figure the DNA to be sequenced is
enclosed within the box. Note that
unless the primer is also labelled with
a radioisotope the smallest band with
the sequence CGTAAGG
dC
will not be
detected by autoradiography, as no
labelled bases were incorporated.
Direction of
electrophoresis
