Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

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Figure 8.10
The structure of α- 32 P-deoxyadenosine triphosphate
([α- 32 P]dATP).
(a) Labeling by nick translation
DNA Pol I
+ 32 P-dATP
(c) Labeling by random priming
O – O – O –
– O P O P O P O CH2
O O O
Radioactive 32 P
C
H
(b) Labeling by end-filling
H
OH
O
HC
H
C C
H
Klenow fragment
+ 32 P-dATP
Nicks Labeled regions EcoRI sticky end
Labeled end
Double-stranded
DNA probe
Denature
by heating
Figure 8.11
Methods for labeling DNA.
Part I The Basic Principles of Gene Cloning and DNA Analysis
The probe must now be labeled with a radioactive or other type of marker, denatured
by heating, and applied to the membrane in a solution of chemicals that promote nucleic
acid hybridization (Figure 8.9c). After a period to allow hybridization to take place, the
filter is washed to remove unbound probe, dried, and the label detected in order to
identify the colonies or plaques to which the probe has become bound (Figure 8.9d).
Labeling with a radioactive marker
A DNA molecule is usually labeled by incorporating nucleotides that carry a radioactive
isotope of phosphorus, 32 P (Figure 8.10). Several methods are available:
l Nick translation. Most purified samples of DNA contain some nicked molecules,
however carefully the preparation has been carried out, which means that DNA
polymerase I is able to attach to the DNA and catalyze a strand replacement
reaction (Figure 8.11a). This reaction requires a supply of nucleotides: if one
of these is radioactively labeled, the DNA molecule will itself become labeled.
Nick translation can be used to label any DNA molecule but might under some
circumstances also cause DNA cleavage.
l End filling is a gentler method than nick translation and rarely causes breakage of
the DNA, but unfortunately can only be used to label DNA molecules that have
sticky ends. The enzyme used is the Klenow fragment (p. 49), which “fills in” a
sticky end by synthesizing the complementary strand (Figure 8.11b)). As with nick
translation, if the end filling reaction is carried out in the presence of labeled
nucleotides, the DNA becomes labeled.
Single-stranded
DNA
Add random
hexamer oligonucleotides
Some hexamers
base-pair
Klenow fragment
+ 32 P-dATP
N
N
C
C
C
NH 2
C
N
Labeled
DNA
N
CH