Norman C. Nelson

Chemiluminescent DNA probes: A comparison of the acridinium ester and dioxetane detection systems and their use in clinical diagnostic assays

Nucleic acid hybridization has the potential to markedly improve the diagnosis of infectious and genetic diseases. Recently, chemiluminescent hybridization assays using acridinium esters and stabilized dioxetanes have been described with sensitivities comparable to those obtained with radioactive labels. Acridinium esters are used as direct labels that are attached to the probe throughout the hybridization reaction. Methods have been developed for labeling DNA probes with acridinium esters at high specific activity and for stabilizing the label under the relatively harsh conditions of hybridization reactions. The label does not affect the kinetics of the hybridization reaction or the stability of the resulting hybrid. The label emits light upon exposure to alkaline peroxide; thus, the assay format can be an extremely simple one. The acridinium ester labels are stable in storage and exhibit extremely rapid light-off kinetics which permit reading large numbers of samples within a brief period as well as limiting the contribution of background signal. A special property of acridinium esters allows chemical destruction of the label when it is present on unhybridized probe, whereas the label is stable to this process when the probe is hybridized. This behavior forms the basis of techniques to minimize assay background signals and allows a homogeneous assay format which does not require physical separation of hybridized and unhybridized probe. The adamantyl-stabilized 1,2-dioxetanes have been used to produce high-sensitivity detection systems for clinical assays. The probe is labeled with enzymes such as alkaline phosphatase or beta-D-galactosidase that hydrolyze the dioxetane derivative to produce a chemiluminescent molecule. As with other enzyme-based labeling systems, the signal increases with time, allowing greater sensitivity to be achieved with longer incubations. The amount of light generated is sufficient to expose sensitive photographic film with extended incubation; therefore, convenient assay formats not requiring instrumentation can be used. Excellent analytical sensitivities have been reported, and by using labels with different light emissions and/or different enzymes on the probes, it is possible to distinguish multiple target sites within a single assay. Because the label is suited for use with solid supports such as polyacrylamide gels, membrane filters, or microscope slides, applications include DNA sequencing, dot and Southern blot hybridizations, and in situ hybridization.

Rapid detection of genetic mutations using the chemiluminescent hybridization protection assay (HPA): overview and comparison with other methods.

The detection of genetic mutations is of paramount importance for the study, diagnosis, and treatment of human genetic disease. Methods of detection generally fall into one of two categories: those to scan for unknown mutations and those to detect known mutations. This review focuses on methods for the detection of known mutations. The hybridization protection assay (HPA) is described in detail. The HPA method utilizes short oligonucleotide probes covalently labeled with a highly chemiluminescent acridinium ester (AE). The assay format is completely homogeneous, requiring no physical separation steps, and can rapidly and sensitively detect all single-base mismatches as well as multiple mismatches, insertions, deletions, and genetic translocations. When very low copy number targets are assayed, HPA is coupled with transcription-mediated amplification (TMA), an isothermal method that amplifies DNA or RNA targets. Other methods that are described for the detection of known mutations include hybridization with sequence-specific oligonucleotides, hybridization to oligonucleotide arrays, allele-specific amplification, ligase-mediated detection, primer extension, and restriction fragment analysis. The advantages and limitations of each of these methods are discussed. Methods to scan for unknown mutations are briefly described.