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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. 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

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  1. Making and Using an Oligo Probe Labeled withAlkaline Phosphatase Alk-Phos Direct Amersham Life Technologies

  2. 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

  3. 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!

  4. 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

  5. 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.

  6. Lysine Arginine Histidine

  7. Note available amino group on each of the bases adenine and cytosine.

  8. Formaldehyde crosslinking Protein Formaldehyde Schiff base or imine A or C of Nucleic Acid oligo- or polymer

  9. 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

  10. 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.

  11. 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!

  12. 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

  13. Excited anion Light producing reaction [2’spiroadamantane]-4-methoxy-3-[3”-(phosphoryl)phenyl]1,2,-dioxetane

  14. 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

  15. 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

  16. 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

  17. 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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