Chemiluminescence and Bioluminescence, Analysis by
531
4.4
Reporter Genes
The
chloramphenicol
acetyltransferase
gene (CAT) is used extensively as a reporter
gene, but assay of the gene product is slow
and requires the use of tritiated reagents.
alcohol
+
NAD
+
alcohol dehydrogenase
−−−−−−−−−−−−−→
acetaldehyde
+
NADH
NADH
+
FMN
NAD
(
P
)
H:FMN
oxidoreductase
−−−−−−−−−−−→
NAD
+
+
FMNH
2
FMNH
2
+
decanal
+
O
2
marine bacterial
luciferase
−−−−−−−−−−→
FMN
+
H
2
O
+
decanoic acid
+
light
(
1
)
glucose
+
O
2
glucose oxidase
−−−−−−−−−→
gluconolactone
+
H
2
O
2
H
2
O
2
+
luminol
peroxidase
−−−−−−→
3-aminophthalic acid
+
N
2
+
2H
2
O
+
light
(
2
)
The gene for ±refly luciferase (
luc
)h
a
s
been cloned and is an effective alternative
Fig. 6
Charge-coupled device camera
image of a chemiluminescent DNA
sequencing blot (M13mp18
single-stranded DNA). The image was
obtained using a Photometrics Star I
CCD camera. Biotinylated DNA
sequencing reactions were loaded onto
the gel in the following order (left to
r
ight)
:A
,C
,G,T
.CLdetect
ionofbound
streptavidin–alkaline phosphatase was
performed using CSPD
.(Courtesyof
Chris Martin, Tropix Inc., Bedford, MA.)
to CAT as a reporter gene for studying
transcriptional activity of cloned genomic
sequences. Luciferase activity in trans-
fected cells is measured by lysing the cells
with a detergent (e.g. Triton X-100), adding
ATP + Mg
2
+
, injecting ±refly luciferin,
and measuring the flash of light (detection
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