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

Making and Using an Oligo Probe Labeled withAlkaline 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 Visualization – light production by action of

the enzyme alkaline phosphatase on the substrate CDP-Star

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

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 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

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

Formaldehyde crosslinking

Protein Formaldehyde

A or C of Nucleic Acid oligo- or polymer

Schiff base or imine

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 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)

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

Excited anion

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)

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 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.

10-3 = milli 10-6 = micro 10-9 = nano 10-12 = pico 10-15 = femto 10-18 = ??? 10-21 = zepto