Page 270 - Color Atlas of Biochemistry

Genetic engineering 261

A. Gene libraries

Imprint on
Host cell plastic membrane
Gene library
in phages
Gene probe

Plate
with phage plaques Hybrid-
ization
Positive plaque

Washing,
detection
of the
gene probe

DNA Gene Gene probe
isolation
Restriction Phage
Cloned gene repli-
cation Membrane

B. Sequencing of DNA

5' 3' GA T C
a
3' 5' Protein
sequence
Synthetic primer Single-stranded preparation
Hybridization DNA
5' sequence
b
3'
3' N
C
c T Phe
T
G A T C C
A Tyr
+ 4 dNTP + 4 dNTP + 4 dNTP + 4 dNTP T
+ ddGTP + ddATP + ddTTP + ddCTP T
+ Polymerase + Polymerase + Polymerase + Polymerase C Ala
G
T
G A T C C Thr
Example: G
A
= ddG
A
A Glu
G
A
A Glu
G
G
d Gel electrophoresis
T Met
e Visualization of the fragments
A
f Reading off ( ) : ACGATG ... 5' N
1. Method 2. Sequence pattern

Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme
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