Maxim-Gilbert Sequencing

32
Detection: 5'- P-labeling

A. Cleavage at the 3'-side of guanosines with a) dimethylsulfate b) , c) piperidine.

32
P O 32 H3 C O 32
P P
N N
NH + NH
O
O O N N NH2 H3 CO S OCH3 O O N N NH2 O O
OH
O H2 O, ∆

O O H3 C O O
O O O
- P - P N - P
O O O O lose NH O O

N N NH2

32 32 32
P P P

O OH H O OH H O OH H
O N + N+

O H H
O H
O O O-
P P H O
-
-
O O O O N -
P
O O

B. Cleavage at the 3'-side of adenosines and guanosines with a) dimethyl sulfate b) HCO2H, c)
piperidine.
32 NH2 NH2
P 32
P
32
P
N N
N N
O +
O O N N H3 CO S OCH3 O O N N O O
HCO 2 H, H2O OH
O CH3
NH2
O O O
O O N O
- P - P N - P
O O O O lose + O O
N N
H
CH3

32 32 32
P P P

O OH H O OH H O OH H
O N + N +

O H H
O O H
O -
P P H O
- -
O O N O
O O
- P
O O
C. Cleavage at the 3'-side of thymidines and cytidines with a) hydrazine, b) , c) piperidine.
32
32 H
+ P
32
P O
P O NH2
HN H
HN O N NH
O CH3
O CH3 O N
O N O
O N H2 N NH2 O
O O
HN NH2
H2 O O
O O
O O P
O - P -
O O
- P O O
O O

32 32
P P

O O
O OH NH2 O OH NH2
N N

O H H
O H -
O
P H O
-
O O N -
P
O O

D. Cleavage at the 3'-side of cytidines with a) hydrazine, 1.5 M NaCl, b) , c) piperidine.

32 NH2
P
32 H+ H+
P NH2 HN
32
P NH2 NH
N O
N
O O N
O O NH
O O N
O N H2 N NH2
O
HN NH2
O
1.5 M NaCl O
O P
O O -
O P O O
-
- P O O
O O

32 32
P P
32
P
NH2 O O
H
O N NH O OH NH2 O OH NH2
O O N N N
NH2
+ O H H
O H
O-
O P H O
O -
O O N P
- P -
O O
O O
Separation and detection of 32P-labeled DNA fragments by polyacrylamide gel electrophosesis (PAGE).
DNA fragments separated based upon charge, which is proportional to the length of the DNA fragment.

G A&G C C&T

G
T
A
A
C
G
T
A
A
T
C
A
C
A
G
 Sanger Sequencing
Enzymatic replication of the DNA fragment to be sequnced with DNA polymerase, Mg(+2), dNTP's,
ddNTP's and 32P-labeled pimers.

ddNTP's
NH2 O

N N NH

O O O O O O
N N N O
-O P O P O P O -O P O P O P O O
O
O- O- O- O- O- O-

ddATP ddTTP

O NH2

N N
NH
O O O N O O O N O
N NH2
-O P O P O P O -O P O P O P O O
O
O- O- O- O- O- O-

ddGTP ddCTP

The extended 32P-labeled primers are separated by PAGE

ddA ddG ddC ddT

C G
A T
T A
T A
G C
C G
A T
T A
T A
A T
G C
T A
G C
T A
C G
 Sanger sequencing with flourescent ddNTP's
H H
O N O NH2 N O

HN H3 C N H3 C
N N
O N O N
-3 O -3
H3 O9 P3 O O
H3 O9 P3 O (CH2 )2 (CH2 )2
O O
H3C CH3

-
O O O- O O O
CH3 CH3 CH3 CH3

ddT
ddC

O O O O
N N
CH3 HN CH3 HN

H3 C CH3 NH2 O

O - N - NH
O O O O O
N N N
-3 -3 N NH2
H3 O9 P3 O H3 O9 P3 O
O O

ddA ddG

Excitation: ~ 490 nM, Emmission: ddT= 526 nm. ddC= 519 nm, ddA= 512 nm, ddG= 505 nm