Antibody-enhanced microplate hybridization assays.

A method for signal enhancement in a colorimetric, nonradioactive and quantitative microplate hybridization assay is described. The signal enhancement provides for the direct detection of viral DNA in serum samples without the use of DNA amplification.

Nonradioactive, colorimetric microplate hybridization assay for detecting amplified human immunodeficiency virus DNA.

A nonradioactive, colorimetric microplate hybridization procedure was used to assay human immunodeficiency virus (HIV) DNA, amplified by the polymerase chain reaction (PCR). Under the PCR conditions used, four proviral copies per 150,000 cells were detected by amplifying a series of DNA mixtures that contained various copy numbers of HIV. Assays of PCR-amplified DNA from peripheral blood mononuclear cells of seronegative individuals yielded negative results (104 of 104), whereas samples from seropositive individuals yielded > 99% positive results (141 of 142). Similar results were obtained in a chemiluminescent assay with an acridinium ester-labeled probe and in a solution hybridization assay in which a 32P-labeled probe was used.

Number and distribution of methylphosphonate linkages in oligodeoxynucleotides affect exo- and endonuclease sensitivity and ability to form RNase H substrates.

Oligodeoxynucleotides with different arrangements of methylphosphonate linkages were examined for nuclease sensitivity in vitro, stability in tissue culture, and ability to form RNase H-sensitive substrates with complementary RNA. After nuclease treatment, resistance was demonstrated by the ability to alter the electrophoretic mobility of a labeled complementary phosphodiester oligodeoxynucleotide. Both 5'- and 3'-exonuclease activities were retarded by methylphosphonate linkages. Methylphosphonate-containing oligodeoxynucleotides with 1-5 adjacent phosphodiester linkages were tested as substrates for the endonucleases DNase I and DNase II. The results indicated that a span of three or fewer contiguous internal phosphodiester linkages led to the greatest resistance to endonuclease. However, in serum-supplemented culture medium half-lives of these oligodeoxynucleotides were independent of the number of contiguous phosphodiester linkages. Methylphosphonate-containing oligodeoxynucleotides were hybridized to RNA runoff transcripts and tested as substrates for RNase H. The results indicated that a span of three internal phosphodiester linkages in the oligodeoxynucleotide was necessary and sufficient to direct cleavage of the RNA in the duplex.

Detection of herpes simplex virus DNA from genital lesions by in situ hybridization.

Lesion specimens from 118 episodes of recurrent genital herpes were used to compare herpes simplex virus (HSV) isolation with a direct specimen test for in situ DNA hybridization utilizing a biotinylated probe. The frequency of detection of HSV was similar with both tests; HSV was isolated from 81% of vesicular lesions, 76% of pustules, and 67% of ulcers, while HSV DNA was detected in 77, 76, and 55% of lesions in these stages, respectively. Utilizing both methods, HSV was identified in 91, 94, and 79%, respectively. The sensitivity and specificity of the DNA probe in comparison to standard viral isolation in tissue culture were 92 and 63%, respectively. Seven DNA-positive, viral isolation-negative specimens were obtained from patients who had positive culture confirmation at some time subsequent or prior to enrollment, suggesting that these were true positive results. The sensitivity of the DNA probe was dependent on cellular content of the specimen, and 36 (28%) of the 127 submitted specimens had fewer than 20 nonsuperficial cells. The DNA probe was rapid and convenient; its major disadvantage was the lack of type-specific information. The performance of the probe in lower-prevalence populations and in asymptomatic shedding of HSV remains to be evaluated.

Synthesis and hybridization of a series of biotinylated oligonucleotides.

A series of oligonucleotides containing biotin-11-dUMP at various positions were synthesized and compared in quantitative, colorimetric hybridization-detection studies. A deoxyuridine phosphoramidite containing a protected allylamino sidearm was synthesized and used in standard, automated synthesis cycles to prepare oligonucleotides with allylamino residues at various positions within a standard 17-base sequence. Biotin substituents were subsequently attached to the allylamino sidearms by reaction with N-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester. These oligomers were hybridized to target DNA immobilized on microtiter wells (ELISA plates), and were detected with a streptavidin-biotinylated horseradish peroxidase complex using hydrogen peroxide as substrate and o-phenylenediamine as chromogen. We found that the sensitivity of detection of target DNA by biotin-labeled oligonucleotide probes was strongly dependent upon the position of the biotin label. Oligonucleotides containing biotin labels near or off the ends of the hybridizing sequence were more effective probes than oligonucleotides containing internal biotin labels. An additive effect of increasing numbers of biotin-dUMP residues was found for some labeling configurations.

Immunodetection with streptavidin-acid phosphatase complex on Western blots.

A technique for the detection of nanogram amounts of protein blotted onto nitrocellulose membranes has been developed using nonradioactive probes. Protein transferred to nitrocellulose membranes is detected by a specific antibody followed by incubation with biotinylated anti-antibody. After addition of streptavidin-acid phosphatase complex, incubation with fast violet B salt produces sharp magenta bands. This method allows detection of bands containing less than 20 ng of protein. The procedure does not use radioactive or carcinogenic materials.

Comparison of radio-labeled DNA probe with a nonisotopic probe for assay of serum hepatitis B virus DNA.

A biotin-labeled DNA probe was compared to a 32P radio-labeled DNA probe for the detection of serum hepatitis B virus (HBV) DNA. Serum specimens were treated with proteolytic enzyme and detergent. DNA was extracted using phenol, denatured in sodium hydroxide and applied to a nitrocellulose filter paper using a vacuum filter device. The nitrocellulose filters were then incubated with either the biotin-labeled or the radio-labeled probe. Annealing of the probe, indicating the presence of HBV-DNA in the sample, was detected either by autoradiography for the 32P-labeled probe or by measuring the presence of an acid phosphatase attached to a streptavidin molecule for the biotin-labeled probe. Using the same 2-day time to complete the assays, excellent correlation of the qualitative and semiquantitative measurements were obtained using 20 HBsAg-positive and 9 HBsAg-negative sera. The nonisotopic assay detected 1.0 pg of HBV-DNA, a sensitivity comparable to reported sensitivities of 32P-labeled HBV-DNA probes when similar assay times are used. 0.02 pg/microliter of HBV-DNA was detected in a normal serum to which HBV-DNA in a recombinant plasmid was added. Our results indicate that the biotin-labeled HBV-DNA probe is approximately as sensitive as the radio-labeled probe for the detection of HBV-DNA using a similar assay time. Isotopic probe assays are more sensitive with longer assay times. The biotin-labeled probe offers the advantage of a longer shelf life and a nonisotopic assay procedure.

Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties.

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.

Replication of poly dA and poly rA by a drosophila DNA polymerase.

The activity of a 7.3S-8.3S Drosophila DNA polymerase was characterized in detail using poly dA.p(dT)[unk] and poly rA.p(dT)[unk]. With poly dA.p(dT)[unk], Mg(2+) ion was the preferred divalent cation, and enzyme activity was inhibited by K(+) ion and by spermidine. With poly rA.p(dT)[unk], Mn(2+) ion was the preferred divalent cation and enzyme activity was stimulated by K(+) ion and by spermidine. The dependence of enzyme activity on the concentration of primer-template and on the ratio of primer to template was the same in both reactions. The two enzyme activities were identically inhibited by N-ethylmaleimide. Poly dA was replicated extensively and poly rA was replicated partially. The activation energy for poly dA replication was twice that for poly rA replication. Enzyme activity with poly dA.p(dT)[unk] was more stable to thermal inactivation than was enzyme activity with poly rA.p(dT)[unk]. These studies suggest that the same enzyme responds to both the deoxy- and the ribohomopolymer template but that the mechanisms of replication may be different.

Multiple forms of Drosophila embryo DNA polymerase: evidence for proteolytic conversion.

The DNA polymerase in crude extracts of Drosophila melanogaster embryos sedimented at 9.0, 7.3, and 5.5 S on glycerol velocity gradients. The relative proportions of these enzymes depended on the method used to prepare the extract. Extracts of whole embryos contained the 7.3S and the 5.5S DNA polymerases and extracts of dechorionated embryos contained the 9.0S and 7.3S DNA polymerases. The porportion of the 5.5S DNA polymerase increased relative to the 7.3S DNA polymerase during storage of the extract of whole embryos. The protease inhibitor, phenylmethanesulfonyl fluoride, inhibited the formation of the 5.5S DNA polymerase, suggesting that it was proteolytically produced from the 7.3S DNA polymerase. This was demonstrated directly by converting the 7.3S DNA polymerase to the 5.5S DNA polymerase by treatment in vitro with trypsin. The degradation of the enzyme occurred without significant loss of DNA polymerase activity. It is further demonstrated that endogenous proteolysis reduced the chromatographic heterogeneity of the Drosophila DNA polymerase on diethylaminoethyl-Sephadex. When endogenous proteolysis was reduced, three forms of DNA polymerase were isolated by diethylaminoethylcellulose chromatography; two of these enzymes sedimented at 7.3S and the third sedimented at 9.0S. These results demonstrate the physical heterogeneity of the Drosophila DNA polymerase and suggest its similarity to vertebrate DNA polymerase-alpha.