Making and using an oligo probe labeled with alkaline phosphatase
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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.


Lysine

Arginine

Histidine



Formaldehyde crosslinking cytosine.

Protein

Formaldehyde

Schiff base or imine

A or C of Nucleic

Acid oligo- or polymer


Hyb and rinse considerations
Hyb and rinse considerations cytosine.

  • 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 cytosine.

  • 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) cytosine.

  • 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: cytosine.

  • 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 anion cytosine.

Light producing reaction

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


Dioxetane substrates
Dioxetane substrates cytosine.

  • 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) cytosine.

  • 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 cytosine.

  • 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


P.S. cytosine.

  • 10-3 = milli

  • 10-6 = micro

  • 10-9 = nano

  • 10-12 = pico

  • 10-15 = femto

  • 10-18 = ???

  • 10-21 = zepto


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