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Epicentre Forum 3 (1)
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Thermolabile Uracil N-Glycosylase (UNG) hydrolyzes
the N-glycosidic bond between the deoxyribose sugar and uracil
in DNA containing deoxyuridine in place of thymidine. When performing
sequential reactions, it may be advantageous to heat inactivate the uracil N-glycosylase
prior to subsequent applications. Although much of the activity of E.
coli UNG is lost upon heat treatment at high temperatures, inactivation
is not complete and the enzyme appears to renature under conditions used
for other applications. HK™-UNG is fully active at 50°C. However,
it is easily inactivated by brief heat treatment and this inactivation
is irreversible.
To demonstrate this, the activity of HK-UNG was assayed
by measuring the degradation of dU-containing DNA. In the presence of
active HK-UNG, uracil is removed from dU-containing DNA and the resulting
abasic DNA is degraded upon heating. If the HK-UNG is inactive, dU-DNA
remains intact.
HK-UNG was heat-treated in a buffer solution at either
70°C or 95°C to inactivate the enzyme. To test for the ability
of the enzyme to renature following heat treatment, the enzyme was incubated
at room temperature for either one or 16 hours. HK-UNG activity was then
assayed by incubation with a dU-containing DNA at 37°C for 30 minutes,
followed by 70°C for 10 minutes. The samples were electrophoresed
on a 1.4% agarose gel and the gel was stained with ethidium bromide.
As shown in lanes 3-6 in Figure 1, brief heat treatment
of HK-UNG at 70°C or 95°C completely eliminated the activity
of HK-UNG and this inactivation was irreversible. In other experiments,
we have demonstrated that incubation at 65°C also inactivates HK-UNG.
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Figure 1. Irreversible inactivation of HK-UNG. Reactions
were performed as described in the text and the samples were
loaded as shown below. The presence of the band in lanes
3, 4, 5, and 6 demonstrates that HK-UNG activity was not
restored following incubation to promote renaturation. |
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Lane |
HK-UNG |
Heat Treatment |
Renaturation Times |
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M |
DNA Markers |
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1 |
- |
none |
0 hr. |
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2 |
+ |
none |
0 hr. |
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3 |
+ |
70°C, 10 min |
1 hr. |
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4 |
+ |
70°C, 10 min. |
16 hr. |
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5 |
+ |
95°C, 10 min. |
1 hr. |
|
6 |
+ |
95°C, 10 min. |
16 hr. |
|
7 |
- |
none |
0 hr. |
|
M |
DNA Markers |
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