Making and Using an Oligo Probe Labeled with Alkaline Phosphatase

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 = 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

3. Probe must be labeled in some way so that light can be produced to expose film. Suppose you wanted to determine whether a PCR product is positive for a 14;18 translocation. (or non-radioactive)

4. The basics of our probe system • 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!

5. Cross-link molecule

6. Note: the enzyme undergoes no net change, but the substrate is changed to yield products and light.

7. 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

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

9. 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.

10. Lysine Arginine Histidine Note: the reactive group is in the uncharged state.

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

12. 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

13. 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 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.

14. 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 all hybrids between target and probe!

15. The light producing reaction: • Uses dioxetane substrates • 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!

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

17. 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

18. 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

19. 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 samples in different lanes = more accurate relative quantitation

20. P.S. • 10-3 = milli • 10-6 = micro • 10-9 = nano • 10-12 = pico • 10-15 = femto • 10-18 = atto • 10-21 = zepto