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Making and Using an Oligo Probe Labeled with Alkaline Phosphatase. Alk-Phos Direct Amersham Life Technologies. Outline. Basic idea of the labeled probe The probe labeling reaction = linking of an oligonucleotide to the enzyme alkaline phosphatase Hybridization and rinse considerations

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making and using an oligo probe labeled with alkaline phosphatase

Making and Using an Oligo Probe Labeled withAlkaline Phosphatase

Alk-Phos Direct

Amersham Life Technologies

outline
Outline
  • Basic idea of the labeled probe
  • The probe labeling reaction = linking of an oligonucleotide to the enzyme alkaline phosphatase
  • Hybridization and rinse considerations
  • Visualization – light production by action of the enzyme alkaline phosphatase on the substrate CDP-Star
the basics
The basics
  • The enzyme alkaline phosphatase (alk phos) can produce light given 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!
the labeling reaction
The labeling reaction
  • Oligonucleotide or polynucleotide probe
  • Alkaline phosphatase enzyme
    • specially developed thermostable enzyme
      • thermostability allows a broader range of temperatures for establishing appropriate hybridization stringency
  • Formaldehyde crosslinker
chemistry of the formaldehyde cross linking reaction
Chemistry of the formaldehyde cross-linking reaction
  • Proteins can be covalently cross-linked to nucleic acids by formaldehyde.
    • Formaldehyde can also cross-link proteins to each other.
  • 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.
slide8
Lysine

Arginine

Histidine

slide10
Formaldehyde crosslinking

Protein

Formaldehyde

Schiff base or imine

A or C of Nucleic

Acid oligo- or polymer

hyb and rinse considerations
Hyb and rinse considerations
  • 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 considerations1
Hyb and rinse considerations
  • 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.
      • Denaturant  lowered Tm, so inclusion of a denaturant means you can lower the temperature and thereby preserve enzyme activity.
      • Urea is less damaging to AlkPhos than formamide, the traditional denaturant in hybridization solutions.
hyb and rinse considerations cont d
Hyb and rinse considerations (cont’d)
  • 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
  • Short rinses required to avoid losing hybrids between target and probe!
the light producing reaction
The light producing reaction:
  • Occurs in alkaline conditions
    • Caution: Low pH will
      • inhibit alkaline phosphatase enzyme activity.
      • reverse the cross-links formed during the formaldehyde driven cross-linking reaction!
  • Uses dioxetane substrates
light producing reaction
Excited anionLight producing reaction

[2’spiroadamantane]-4-methoxy-3-[3”-(phosphoryl)phenyl]1,2,-dioxetane

dioxetane substrates
Dioxetane substrates
  • can detect < 100 fg of nucleic acid in a single band
    • radioactivity 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
Dioxetane substrates (cont’d)
  • 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
  • AMPPD, CSPD, CDP- Star don’t work with nitrocellulose
    • Nitrocellulose is insufficiently hydrophobic
cdp star
CDP-Star
  • is a stabilized dioxetane
  • has short lag phase  fast results
    • The turnover rate for various enzyme/substrate combinations varies. The higher the turnover rate, the shorter the lag phase.
      • Turnover rate = the number of enzymatic reaction repetitions/unit time
  • yields maximum light by 4 hours and continues light production for several days
    • allows multiple exposures to film, so the user
      • can optimize signal to noise
      • can more accurately compare intensities of sample in different lanes = more accurate relative quantitation
slide19
P.S.
  • 10-3 = milli
  • 10-6 = micro
  • 10-9 = nano
  • 10-12 = pico
  • 10-15 = femto
  • 10-18 = ???
  • 10-21 = zepto
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