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

2. Outline n Basic idea of the labeled probe n The probe labeling reaction = covalent linking of an oligonucleotide to the enzyme alkaline phosphatase n Hybridization and rinse considerations dictated by the nature of the probe n Visualization – light production by action of the enzyme alkaline phosphatase on the substrate CDP-Star

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

4. The basics of our probe system n The enzyme alkaline phosphatase (alk phos) can produce light from reaction on an appropriate substrate. n Alk phos can be covalently linked to a nucleic acid probe and remain active. n The probe labeled with alk phos can hybridize to target DNA on a membrane. n The alk phos stays active even after hybridization. n 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 n Oligonucleotide or polynucleotide probe n Alkaline phosphatase enzyme u specially developed thermostable enzyme F thermostability allows a broader range of temperatures for establishing appropriate hybridization stringency n Formaldehyde crosslinker

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

9. Chemistry of the formaldehyde cross-linking reaction n Proteins can be covalently cross-linked to nucleic acids by formaldehyde. u Formaldehyde can also cross-link proteins to each other. n Formaldehyde is a highly reactive dipolar compound. n Carbon atom of formaldehyde acts as nucleophilic center. n Amino or imino group + formaldehyde  Schiff base n Schiff base intermediate + 2nd amino group  cross-link n 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 n The presence of AlkPhos interferes with base pairing u 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 u i.e., the presence of Alk Phos has lowered the Tm of the probe. F Think of needing a new mathematical term in the Tm equation

13. Hyb and rinse considerations n AlkPhos Direct hybridization and 1 o wash solutions contain urea, a denaturant. Why? u Background: You would like to be able to hybridize at a temperature low enough to preserve the activity of the Alk Phos enzyme. F Denaturant  lowered Tm, so inclusion of a denaturant means you must lower the temperature. The lowered temperature helps to preserve enzyme activity. F Urea is less damaging to AlkPhos than formamide, the traditional denaturant in hybridization solutions.

14. Hyb and rinse considerations (cont’d) n At or near the Tm, a perfectly complementary oligonucleotide is essentially completely bound, or completely free (no bubbles in the hybrid). u During hybridization, in high [probe], when an oligonucleotide separates from the target, it can be replaced by another probe u During rinse, in the absence of additional probe, when an oligonucleotide separates from target, it won’t be replaced by another probe n Short rinses required to avoid losing all hybrids between target and probe!

15. The light producing reaction: n Uses dioxetane substrates n Occurs in alkaline conditions u Caution: Low pH will F inhibit alkaline phosphatase enzyme activity. F reverse the cross-links formed during the formaldehyde driven cross-linking reaction!

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

17. Dioxetane substrates n can detect < 100 fg of nucleic acid in a single band u radioactivity is still more sensitive n half-life of excited molecule ranges from 2 minutes - several hours - several days u depends on specific dioxetane molecule and environment in which the excited molecule is found

18. Dioxetane substrates (cont’d) n nylon membranes stabilize decay u excited anion stabilized by hydrophobic pocket u hydrophobic interactions  blue shift to 466 nm F chlorinated dioxetanes (CSPD) minimize both hydrophobic interactions and self-aggregation to cause more rapid decay n AMPPD, CSPD, CDP- Star don’t work with nitrocellulose u Nitrocellulose is insufficiently hydrophobic

19. CDP-Star n is a stabilized dioxetane n has short lag phase  fast results u The turnover rate for various enzyme/substrate combinations varies. The higher the turnover rate, the shorter the lag phase. F Turnover rate = the number of enzymatic reaction repetitions/unit time n yields maximum light by 4 hours and continues light production for several days u allows multiple exposures to film, so the user F can optimize signal to noise F can more accurately compare intensities of samples in different lanes = more accurate relative quantitation

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