A new approach to the isolation of RNA-DNA hybrids and its application to the quantitative determination of labeled tumor virus RNA

A procedure has been developed by which the hybrid formed between a labeled RNA and complementary DNA can be selectively separated from all other single and double-stranded nucleic acids. We describe the application of this procedure to the quantitative determination of labeled avian tumor virus RNA. Purified DNA complementary to avian myeloblastosis virus RNA is extended at its 3′ terminus with 40 to 60 dCMP residues, using terminal deoxynucleotidyl-transferase. The elongated DNA is annealed with the labeled nucleic acid preparation and the mixture is passed through a column of Sephadex to which poly(I) has been covalently bound. The poly(I) retains the specific RNA-DNA hybrids by virtue of their poly(C) extension. The column is washed with RNAase to degrade nonhybridized RNA, the RNA retained on the column is eluted with formamide and its radioactivity is determined. The background hybridization was reduced to 0.003 to 0.008% by addition of oligo(C)_5.20 to the hybridization mixture and by carrying out the adsorption to the poly(I)-Sephadex column in the presence of poly(U). The hybridization efficiency was about 50%. The content of radioactive Rous sarcoma virus-specific RNA was determined in infected and uninfected cells after labeling with [³H]uridine for two hours. The content of labeled virus-specific RNA in infected cells was 0.6 to 0.9% and 0.05% in uninfected cells. The value found for monkey cell RNA was 0.009%. This method can be used for the detection of hybrids between labeled RNA and complementary DNAs too short to allow quantitation by conventional methods. If the RNAase step is omitted the procedure can be used for the isolation of any RNA for which a complementary DNA is available, as well as for its precursor.     

DNA hybridization biosensors using polylysine modified SPCEs

Two electrochemical DNA hybridization biosensors (genosensors) for the detection of a 30-mer sequence unique to severe acute respiratory syndrome (SARS) virus are described in this work. Both genosensors rely on the hybridization of the oligonucleotide target with its complementary probe, which is immobilized on positively charged polylysine modified screen-printed carbon electrodes (SPCEs), through electrostatic interactions. In one design, a biotinylated target is used and the detection of the hybridization reaction is monitored using alkaline phosphatase labeled streptavidin (S-AP). This enzyme catalyzes the hydrolysis of the substrate 3-indoxyl phosphate (3-IP) to indigo, which is then solubilized to indigo carmine and detected by means of cyclic voltammetry (CV). In the other design, the target is labeled using an Au(I) complex, sodium aurothiomalate, and the duplex formation is detected by measuring, for first time, the current generated by the hydrogen evolution catalyzed by the gold label. Using 30 min of hybridization time, a detection limit of 8 pM is calculated for the enzymatic genosensor. Although this good sensitivity cannot be reached with the metal label (0.5 nM), the use of this label allows a considerable decrease of the analysis time. Both genosensors do not require the modification of the oligonucleotide probe and using stringent experimental conditions (60 min of hybridization time and 50% formamide in the hybridization buffer) can discriminate between a complementary oligonucleotide and an oligonucleotide with a three-base mismatch.     

DNA hybridization sensor based on aurothiomalate electroactive label on glassy carbon electrodes

In this work, a gold complex is used as electroactive label for monitoring hybridization assays on glassy carbon electrodes. Ionic gold is bound to a 30-mer sequence of the SARS (severe acute respiratory syndrome) virus, responsible for the atypical pneumonia, using sodium aurothiomalate. In order to label this single strand, a mixture of sodium aurothiomalate and the strand is prepared. Then, it is incubated for 24 h at 37 degrees C and, finally, free gold is separated from the labeled strand by a dialysis against a 0.15M NaCl solution (pH 7.5). The DNA hybridization sensor is designed immobilizing the complementary probe on the pre-treated electrode surface and, then, the hybridization reaction takes place with the gold labeled strand. The electrochemical determination is based on the catalytic effect of electrodeposited gold on the reduction of silver ions. In non-stringent experimental conditions, a limit of detection of 15 fmol (30 microL) is obtained, and discrimination between a complementary oligonucleotide and a three-based mismatch complementary oligonucleotide is achieved. For the discrimination of a single-base mismatch, is needed to use stringent conditions (50% of formamide in the hybridization buffer).     

Cytochemical hybridization with fluorochrome-labeled RNA. II. Applications

The cytochemical detection of specific DNA sequences by hybridization with fluorochrome-labeled RNA and detection of the hybrids by fluorescence microscopy is described. RNAs complementary to the DNA of the kinetoplasts of Crithidia luciliae (an insect trypanosome) or to adenovirus-5 (Ad-5) DNA were labeled with the hydrazine derivative of tetramethylrhodamine isothiocyanate (TRITC). The specificity of the reactions between the complementary RNAs labeled both with 3H and tetramethylrhodamine was studied by cross-hybridization experiments using a model system in which the DNAs were bound to Sepharose beads. The extent of the reaction was measured by scintillation counting of the bead suspensions and quantitative fluorescence microscopy of individual Sepharose beads. The ability of the rhodamine-labeled cRNAs to hybridize and the absence of interference of the fluorochrome label with the specificity of the hybridization reaction was thus demonstrated. After cytochemical hybridization on microscopic preparations of C. luciliae cells the rhodamine-labeled kinetoplast cRNA stains only the kinetoplasts. No fluorescence was observed in the nuclei. After cytochemical hybridization of rhodamine-labeled Ad-5 cRNA with virus infected KB cells a distinct staining pattern in the nuclei was observed. No fluorescence was seen in uninfected cells, or after hybridization with heterologous rhodamine-labeled RNA. The possibilities and limitations of cytochemical hybridization with rhodamine-labeled RNA are discussed.     

Oligonucleotide quartz crystal microbalance sensor for the microalgae Alexandrium minutum (Dinophyceae)

We report the immobilization on a gold surface of a 20-base DNA probe labeled with disulfide group and on the selective hybridization with the complementary 20-base DNA strand. The oligonucleotide probe is the complementary strand of a partial sequence of the gene encoding for a large ribosomal RNA sub-unit which is a coding sequence of Alexandrium minutum DNA, a microalgae that produces neurotoxins responsible for paralytic shellfish poisoning on European and Asian coasts. The kinetics of DNA probe immobilization and hybridization were monitored in situ by using a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the setup is stable to within a few hertz, corresponding to the nanogram scale, for 3h and makes it possible to follow frequency change from immobilization of the probe to hybridization of the complementary DNA target. This setup constitutes a biosensor, which is sensitive and selective, and the hybridization ratio between hybridized complementary DNA and immobilized DNA probes is 47%.     

In Situ Hybridization at the Electron Microscopic Level Using a Bromodeoxyuridine Labeled DNA Probe

A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level. A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells. After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM). and others were embedded in Quetol for electron microscopy (EM). The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid. In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum. Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level. Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here. This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues.     

间接核酸杂交方法的建立

建立了一种新的核酸杂交手段一间接核酸杂交方法,其突出的优点是用一种共同的核酸标记物就可检查不同的基因组或不同的基因。我们重组乙型肝炎病毒或EB病毒的核酸片段于噬菌体M_(13)mp8载体,以此重组的单链DNA为第一夹心层,用~(32)P标记的双链噬菌体DNA作为共同探针,检查乙型肝炎病毒和EB病毒的核酸,获得满意的结果。应用该法进行细胞内的原位杂交,检查细胞内存在的EB病毒基因效果亦佳。     

Detection of Epstein-Barr virus (EBV) DNA sequences using in situ hybridization.

In situ hybridization was used to detect Epstein-Barr virus (EBV) DNA sequences under conditions where the virus DNA is replicating spontaneously and where it is induced to do so following superinfection. The in situ reaction itself is influenced by several parameters, analogous to conventional nucleic acid hybridization, consideration of which should help to optimize the designing of in situ hybridization reactions in general. Both EBV complementary RNA (cRNA) and EBV DNA synthesized in vitro can efficiently detect the virus DNA sequences in situ. The findings presented here can therefore be utilized in both the study of EBV-cell interactions and, more generally, in studies using in situ hybridization as a general approach.     

New Method of Studying the Precursor-product Relationship between High Molecular Weight RNA and Messenger RNA

THREE principal methods have been used to test whether giant heterogeneous RNA is a precursor to cytoplasmic messenger RNA in animal cells. First, competition-hybridization to DNA has shown that there is certainly a degree of similarity between the two types of molecule^1–5. The conditions used in most of these experiments allowed only the hybridization of the reiterated fraction of DNA or RNA. It is possible therefore that the competing sequences are similar, but not identical and in any case may represent only a small fraction of the total sequences. Scherrer et al.⁶, using conditions which might allow the hybridization of some of the slow sequences of DNA and which would give meaningful competition, conclude that there is a precursor-product relationship between giant heterogeneous RNA and cytoplasmic messenger RNA. Darnell, following the uptake of labelled nucleotides into the two fractions⁷, has shown that a precursor product relationship is possible although not proven. Third, the differential inhibition of synthesis of the two fractions by cordycepin⁸ has been taken to show that the giant heterogeneous RNA cannot be a precursor to the cytoplasmic messenger RNAs. Although these data would confirm previous observations on the rate of uptake and decay of label in nucleoplasmic and cytoplasmic RNA in the presence of actinomycin⁹, this type of experiment is rather indirect and open to other interpretations because the inhibitors might not act exclusively and directly on one step of the overall metabolic pathway. A relationship has been clearly established between high molecular weight precursors and the final RNA produced only for SV40 transformed cells¹⁰. In this case the hybridization experiments were much simplified because SV40 DNA, which is very small in comparison with animal DNA, is readily available. In this communication we describe a direct and general way of approaching the problem which makes novel use of molecular hybridization and can be applied to messenger RNAs which are not easily labelled. The method also overcomes the difficulty of hybridizing to the extremely complex DNA of higher organisms.     

Size and base composition of RNA segments in complementary strands of supercoiled plasmid ColE1 DNA

Preparations of ColEl plasmid DNA synthesized in the presence of chloramphenicol were separated into samples having gaps resulting from removal of ribonucleotides in one or the other of the complementary DNA strands. These samples were used as templates for repair resynthesis reactions using DNA polymerase of Rous sarcoma virus and α-³²P-labeled deoxyribonucleoside 5′-triphosphates. Reactions involved the incorporation of each labeled nucleotide in the presence of three unlabeled nucleotides, and also the incorporation of all four labeled nucleotides followed by complete digestion and electrophoretic separation of the products. By these two methods the RNA integrated in the light strand of ColEl DNA was found to comprise an average of 38 ribonucleotides with a base composition of 17G, 5A, 8C, and 8U. The RNA segment in the heavy strand consists of an average of 15 ribonucleotides with a base composition of 5G, 2A, 4C, and 4U.     

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

Summary A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.     

DNA replication in isolated nuclei : The fate of pulse-labelled DNA subunits

The fate of ³H-dTTP incorporated into DNA in isolated S phase nuclei from Chinese Hamster Ovary cells was examined. ³H-dTTP observed in 4 S DNA subunits after a pulse-label becomes acid-soluble during a chase performed under conditions which permit continued active DNA synthesis. ³H-dTTP incorporated into longer DNA subunits is not affected by these chase conditions. This selective loss of 4 S pulse-label requires active DNA synthesis. In incubations which do not permit continued DNA synthesis, either there is little loss of label or the loss occurs equally from the 4 S and larger DNAs. Possible reasons for a metabolically active 4 S subunit are discussed.     

A cloned 23S rRNA gene fragment of Bacillus subtilis and its use as a hybridization probe of conserved character

Abstract A subclone of plasmid p14B8 containing the major part of a 23S rRNA gene of Bacillus subtilis was constructed and designated pJK1. Labeled plasmid pJK1 could be used as a DNA probe with conserved gene sequences. DNA-DNA hybridization experiments between filter-bound DNA from various bacteria and labeled pJK1 showed a good correlation between oligonucleotide sequence analysis of 16S rRNA and DNA homology values. Application of suboptimal or stringent hybridization conditions and an additional short incubation under the same conditions following hybridization yielded the best data for differentiating organisms related to B. subtilis from less or non-related bacteria.     

Two complementary strand-specific termination sites for adenovirus DNA replication

Adenovirus type 2 DNA, specifically labeled at the termini for DNA replication, was prepared by isolation of viral DNA molecules which were completed during short pulses with ³H-thymidine. The distribution of radioactivity in the two complementary strands at the termini for DNA replication was determined by liquid phase hybridization and gel electrophoresis. At the right-hand terminus, nearly all radioactivity was found in the viral h strand, whereas at the left-hand terminus, most radioactivity was confined to the viral I strand. The results suggest that both molecular ends serve as origins and termini for replication of adenovirus type 2 DNA.     

Synthesis of somatomedin C/insulin-like growth factor I by human placenta

We have reported the presence of insulin-related poly A+RNA sequences in human placenta by RNA to DNA hybridization. In this study we have used a monoclonal antibody to somatomedin C/insulin-like growth factor I (Sm-C/IGF-I) to identify somatomedin-like proteins whose synthesis is directed by placental mRNA. Poly A+RNA from first trimester and term placenta was translated in a cell-free system using micrococcal nuclease-treated reticulocyte-lysate and [³⁵S]methionine as a label. From 2.0×10⁶ cpm of specifically incorporated [³⁵S]methionine labeled protein, an immunoprecipitate with an apparent molecular weight of 14000 represented about 0.1% of total radioactivity in the translational products of poly A+RNA of first trimester placenta. A less prominent band (0.006%) of the same apparent molecular weight was also evident from translational products of term placental mRNAs. This protein could be competed with either acromegalic serum or synthetic Sm-C/IGF-I when added prior to immunoprecipitation. Translational products synthesized from mRNA of term placenta showed a second labeled band of 24000 daltons. This band was less effectively competed by acromegalic serum and not competed with either Sm-C/IGF-I or IGF-II and therefore its identity is uncertain. A protein similar to Sm-C/IGF-I is, therefore synthesized in first trimester placenta and to a lesser extent at term, suggesting developmental changes in Sm-C/IGF-I synthesis. Because Sm-C/IGF-I may act in a paracrine fashion, our findings suggest a role for Sm-C/IGF-I in growth of the placenta during early gestation.     

Multiplex detection of single-nucleotide variations using molecular beacons

We demonstrate that single-nucleotide differences in a DNA sequence can be detected in homogeneous assays using molecular beacons. In this method, the region surrounding the site of a sequence variation is amplified in a polymerase chain reaction and the identity of the variant nucleotide is determined by observing which of four differently colored molecular beacons binds to the amplification product. Each of the molecular beacons is perfectly complementary to one variant of the target sequence and each is labeled with a different fluorophore. To demonstrate the specificity of these assays, we prepared four template DNAs that only differed from one another by the identity of the nucleotide at one position. Four amplification reactions were prepared, each containing all four molecular beacons, but each initiated with only one of the four template DNAs. The results show that in each reaction a fluorogenic response was elicited from the molecular beacon that was perfectly complementary to the amplified DNA, but not from the three molecular beacons whose probe sequence mismatched the target sequence. The color of the fluorescence that appeared in each tube during the course of the amplification indicated which nucleotide was present at the site of variation. These results demonstrate the extraordinary specificity of molecular beacons. Furthermore, the results illustrate how the ability to label molecular beacons with differently colored fluorophores enables simple multiplex assays to be carried out for genetic analysis.     

Complementary strand-specific sequences from unique fragments of adenovirus type 2 DNA for hybridization-mapping experiments

Separation of the complementary strands of adenovirus type 2 DNA by poly(U,G)-CsCl density gradient centrifugation permitted studies of Ad2³ DNA renaturation with independently variable concentrations of each complementary strand. Single-stranded DNA was isolated by hydroxylapatite chromatography following exhaustive incubation under such conditions, and was found to selectively represent sequences of the complement present in excess during the incubation. This result was exploited in a general method for isolation of complementary strand-specific sequences of radioactively labeled Ad2 DNA or restriction enzyme fragments of Ad2 DNA. Liquid phase saturation-hybridization experiments were carried out with labeled DNA representing each complementary strand of the six endo R.EcoRI cleavage fragments of Ad2 DNA and unlabeled messenger RNA prepared from HeLa cells late after productive infections with Ad2. The results were combined with the known endo R.EcoRI cleavage map of Ad2 DNA to construct a low-resolution map showing physically separated regions, on both complementary strands of Ad2 DNA, represented in mRNA late after infection.     

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5′-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5′→3′ exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10–10⁷ initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5′-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.     

The mechanism of dextransucrase action: Biosynthesis of branch linkages by acceptor reactions with dextran

Dextransucrase from Leuconostoc mesenteroides B-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [¹⁴C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [¹⁴C]sucrose. After extensive washings with buffer, the ¹⁴C label remained attached to BGD. This labeled material was previously shown to be [¹⁴C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [¹⁴C]dextran whereas essentially no [¹⁴C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [¹⁴C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [¹⁴C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C₃-OH on an acceptor dextran acts as a nucleophile on C₁ of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.