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.

Retrospective study of Gen-Probe rapid diagnostic system for detection of legionellae in frozen clinical respiratory tract samples.

The Gen-Probe Rapid Diagnostic System for legionellae, which uses 125I-labeled cDNA directed against the rRNAs of legionellae, was evaluated for its ability to detect members of the genus by using clinical specimens which had been frozen at -70 degrees C for 2 to 8 years. Culture and direct immunofluorescence (DFA) results obtained at the time of specimen collection were used to categorize samples. The specimens tested were 112 samples culture positive for legionellae and 230 samples negative on culture and DFA tests. They were tested in a blinded and randomized fashion. Results were expressed in terms of the ratio of counts per minute of the sample to the counts per minute of the provided negative control. A ratio of greater than or equal to 4.0 was picked for optimal specificity. Of the 112 previously positive specimens, 63 (57%) were positive by the probe assay, and of the 230 previously negative samples, 228 (99.1%) were negative. The 51 discrepant specimens were reexamined by culture and DFA testing if adequate amounts remained; this was possible for 34 specimens. On repeat culture, 22 of 33 previously culture-positive samples yielded legionellae and 11 were negative. Ten of the positive repeat cultures yielded two or fewer colonies per plate. One probe-positive but previously culture-negative sample was overgrown by contaminants on repeat culture. Reanalysis of data after exclusion of the 17 unavailable, 11 repeat culture-negative, and 1 unevaluable specimen gave a probe sensitivity of 74% and specificity of 100%. The Gen-Probe test is therefore specific and is of useful sensitivity.

Purification of plasminogen activator from Rous sarcoma virus-infected chick embryo fibroblast culture medium.

A new procedure for the purification of plasminogen activator secreted by cultured Rous sarcoma virus-infected chick embryo fibroblasts was described. The enzyme was isolated from culture medium containing 0.75% calf serum depleted of plasminogen by lysine-agarose affinity column chromatography and of high-molecular-weight protease inhibitors by ultracentrifugation. The culture conditions allowed convenient preparation of large amounts of culture fluid with relatively high concentrations of plasminogen activator. The purification of the enzyme was accomplished by affinity chromatography on fibrin-celite and p-aminobenzamidine-agarose columns, and by gel-filtration chromatography in the presence of urea. The activity was recovered in greater than 90% yield, and the enzyme was essentially homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Yields from 500 ml culture fluid exceeded 500 micrograms.

Synthesis of full-length DNA copies of avian myeloblastosis virus RNA in high yields.

The synthesis of full-length complementary DNA copies of avian myeloblastosis virus RNA is described. The cDNA is estimated by electrophoresis in agarose-methylmercury gels to have a molecular weight of 2.6 X 10(6), equivalent in size to that of the RNA template. Most (60-70%) of the reaction product is the full-length material and yields correspond to 60-70% of the input RNA.

Anticomplementary nature of smaller DNA produced during synthesis of extensive DNA copies of poliovirus RNA.

The reverse transcriptase (RNA-directed DNA nucleotidyltransferase) from avian myeloblastosis virus is able to make an extensive, possibly complete, complementary DNA copy of intact poliovirus RNA. In the presence of high concentrations of deoxyribonucleoside triphosphates, ribonucleoside triphosphates, or sodium pyrophosphate, this DNA is the only species produced. Without these additives, however, a second size class of DNA is also synthesized. This material has a sedimentation coefficient between roughly 4 and 10 S and is produced later in the reaction, largely after synthesis of the larger complementary DNA has ceased. The smaller DNA consists primarily of material anticomplementary to the RNA template and contains a faithful and uniform representation of the viral sequences. It most likely arises by transcription of the larger DNA species.

Synthesis of extensive, possibly complete, DNA copies of poliovirus RNA in high yields and at high specific activities.

The synthesis of large, possibly complete, complementary DNA (cDNA) copies of poliovirus RNA by avian myeloblastosis virus DNA polymerase is described. The cDNA consists of two size classes, the larger of which is approximately 7500 nucleotides. In the presence of excess deoxynucleoside triphosphates, ribonucleoside triphosphates, or sodium pyrophosphate, only the larger material is obtained. Yields of the large cDNA are 50-75% of the input RNA.

A limited number of globin genes in human DNA.

The number of globin genes in human cells was determined by hybridizing DNA from human spleens to (3)H-labeled DNA complementary to human globin mRNA. Assuming the rates of reannealing of complementary DNA and cellular DNA are similar, the extent of hybridization of complementary DNA at various ratios of cellular DNA to complementary DNA indicate that there are fewer than 10 globin gene copies per haploid human genome. An alternative analysis of the data, which introduces no assumptions concerning the relative rates of reaction of complementary DNA and cellular DNA, indicates fewer than 20 globin gene copies are present. DNA isolated from the spleen of a patient with beta(+) thalassemia contained a number of globin gene copies similar to that of normal DNA.

Nucleotide sequence analysis of RNA synthesized from rabbit globin complementary DNA.

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of (32)P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T(1) and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the alpha or beta globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants.

Conditions for using DNA polymerase I as an RNA-dependent DNA polymerase.

Conditions are described for using Escherichia coli DNA polymerase I for synthesizing complementary DNA copies of natural RNA molecules, which are suitable for use in hybridization experiments. The molar ratio of enzyme to template is critical; below a certain level, synthesis is not observed. Hybrids formed with the complementary DNA are of comparable specificity and stability to those formed with complementary DNAs synthesized by viral RNA-directed DNA polymerase. Synthesis of dA-dT polymers, a common occurrence with this enzyme, can be eliminated by including distamycin in the reaction mixture.