Download Making and Using an Oligo Probe Labeled with Alkaline Phosphatase

Making and Using an Oligo
Probe Labeled with
Alkaline Phosphatase
Alk-Phos Direct
Amersham Life Technologies
Outline
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Basic idea of the labeled probe
The probe labeling reaction = covalent
linking of an oligonucleotide to the enzyme
alkaline phosphatase
Hybridization and rinse considerations
dictated by the nature of the probe
Visualization – light production by action of
the enzyme alkaline phosphatase on the
substrate CDP-Star
Suppose you wanted to determine whether a PCR
product is positive for a 14;18 translocation.
(or non-radioactive)
Probe must be labeled in some
way so that light can be
produced to expose film.
The basics of our probe system
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The enzyme alkaline phosphatase (alk phos) can
produce light from reaction on an appropriate
substrate.
Alk phos can be covalently linked to a nucleic acid
probe and remain active.
The probe labeled with alk phos can hybridize to
target DNA on a membrane.
The alk phos stays active even after hybridization.
Addition of substrate to the blot and recording of the
light produced on film shows where on the blot
hybridization occurred!
Cross-link molecule
Note: the enzyme undergoes no net change, but the substrate is
changed to yield products and light.
The labeling reaction
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Oligonucleotide or polynucleotide probe
Alkaline phosphatase enzyme
 specially
developed thermostable enzyme
 thermostability
allows a broader range of temperatures
for establishing appropriate hybridization stringency
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Formaldehyde crosslinker
Formaldehyde crosslinking
Protein
Formaldehyde
Schiff base or
imine
A or C of Nucleic
Acid oligo- or polymer
Chemistry of the formaldehyde
cross-linking reaction
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Proteins can be covalently cross-linked to nucleic
acids by formaldehyde.
 Formaldehyde
can also cross-link proteins to each
other.
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Formaldehyde is a highly reactive dipolar compound.
Carbon atom of formaldehyde acts as nucleophilic
center.
Amino or imino group + formaldehyde  Schiff base
Schiff base intermediate + 2nd amino group 
cross-link
Reaction is reversible at low pH.
Lysine
Arginine
Histidine
Note: the reactive group is in the uncharged state.
Note available
amino group on
each of the bases
adenine and
cytosine.
Hyb and rinse considerations
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The presence of AlkPhos interferes with base pairing
 So,
in any given hybridization solution, probe labeled
with alkaline phosphatase will have more difficulty
hybridizing than a probe labeled with radioactivity or a
less bulky label
 i.e., the presence of Alk Phos has lowered the Tm of
the probe.
 Think
of needing a new mathematical term in the Tm
equation
Hyb and rinse considerations
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AlkPhos Direct hybridization and 1o wash solutions
contain urea, a denaturant. Why?
 Background:
You would like to be able to hybridize at
a temperature low enough to preserve the activity of
the Alk Phos enzyme.
 lowered Tm, so inclusion of a denaturant
means you must lower the temperature. The lowered
temperature helps to preserve enzyme activity.
 Urea is less damaging to AlkPhos than formamide, the
traditional denaturant in hybridization solutions.
 Denaturant
Hyb and rinse considerations
(cont’d)
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At or near the Tm, a perfectly complementary
oligonucleotide is essentially completely bound, or
completely free (no bubbles in the hybrid).
 During
hybridization, in high [probe], when an
oligonucleotide separates from the target, it can be
replaced by another probe
 During rinse, in the absence of additional probe, when
an oligonucleotide separates from target, it won’t be
replaced by another probe
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Short rinses required to avoid losing all hybrids
between target and probe!
The light producing reaction:
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Uses dioxetane substrates
Occurs in alkaline conditions
Caution:
inhibit
Low pH will
alkaline phosphatase enzyme
activity.
reverse the cross-links formed during
the formaldehyde driven cross-linking
reaction!
Light producing reaction
[2’spiroadamantane]-4-methoxy-3-[3”-(phosphoryl)phenyl]1,2,-dioxetane
(1 Substrate)
(3 Products)
Excited anion
Dioxetane substrates
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can detect < 100 fg of nucleic acid in a single band
 radioactivity
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is still more sensitive
half-life of excited molecule ranges from 2 minutes several hours - several days
 depends
on specific dioxetane molecule and
environment in which the excited molecule is found
Dioxetane substrates (cont’d)
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nylon membranes stabilize decay
 excited
anion stabilized by hydrophobic pocket
 hydrophobic interactions  blue shift to 466 nm
 chlorinated
dioxetanes (CSPD) minimize both
hydrophobic interactions and self-aggregation to cause
more rapid decay
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AMPPD, CSPD, CDP- Star don’t work with
nitrocellulose
 Nitrocellulose
is insufficiently hydrophobic
CDP-Star
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is a stabilized dioxetane
has short lag phase  fast results
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The turnover rate for various enzyme/substrate
combinations varies. The higher the turnover rate, the
shorter the lag phase.
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Turnover rate = the number of enzymatic reaction
repetitions/unit time
yields maximum light by 4 hours and continues light
production for several days
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allows multiple exposures to film, so the user
can optimize signal to noise
 can more accurately compare intensities of samples in different
lanes = more accurate relative quantitation
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P.S.
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10-3 = milli
10-6 = micro
10-9 = nano
10-12 = pico
10-15 = femto
10-18 = atto
10-21 = zepto