Study of stringency conditions for human papillomavirus DNA detection on cell lines, frozen and paraffin-embedded tissue sections by in situ hybridization with biotinylated probes

In situ hybridization was mainly used for typing human papillomavirus (HPV) in paraffin-embedded or frozen sections under stringent conditions (SC). We tested 5 different conditions of stringency with biotinylated HPV 1, 2, 16 and 18 probes on 3 cell lines (Sihà and CaSki with HPV16, HeLa with HPV18) by varying the concentration of formamide in the hybridization mixture and washings in order to determine the stringency conditions to be used to assess the presence of HPV and its typing: A-low stringency, hybridization at 35 degrees C below the melting temperature of DNA (Tm-35 degrees C) and washings without formamide; B-low stringency, hybridization and washings at Tm-35 degrees C; C-medium stringency, hybridization at Tm-35 degrees C and washings at Tm-12 degrees C; D-high stringency, hybridization at Tm-12 degrees C and washing without formamide; E-very high stringency, hybridization and washings at -12 degrees C. This study showed that HPV typing required a high stringency. On the contrary, under non stringent conditions (NSC), each cell line was positive with the heterologous probes. When 3 to 5 stringency conditions were assayed on 4 frozen samples, similar results were obtained. Typing required high stringency conditions whereas NSC allowed HPV detection. Furthermore, this study demonstrated the specificity of the reaction in lesions positive with more than one type. Stringent (Tm-12 degrees C) and non stringent (Tm-35 degrees C) conditions of hybridization were further applied to 57 biopsy sections (17 frozen and 40 paraffin-embedded specimens) from typical wart lesions and lesions suspected of HPV.(ABSTRACT TRUNCATED AT 250 WORDS)     

两种DNA探针杂交检测结核分支杆菌方法的研究

为改进结核杆菌DNA探针的特异性与实用性,研制了以生物素标记的两种对结核分支杆菌特异的DNA探针:一个5’端标记的20bp的寡核苷酸探针和一个采用PCR方法合成的188bp长链探针。两种探针分别与结核分支杆菌的全染色体DNA,以及基因组上IS6110序列的一段317bp的PCR扩增产物进行斑点杂交,以碱性磷酸酶(AP)催化的染色反应检测,测试了两个探针的敏感性和特异性。系统地比较研究了两种探针杂交检测条件:探针的浓度选择,杂交温度与洗膜温度的选择,以及杂交与洗膜温度对检测的敏感性与特异性的影响。寡核苷酸探针和188bp探针杂交检测纯化结核分支杆菌基因组DNA的敏感性分别为100ng与6ng,杂交检测PCR产物的敏感性分别是400pg与50pg。两探针的最佳杂交浓度均为40～160ng/ml,最佳杂交温度分别是42℃与68℃,最佳洗膜温度分别是60℃与60～68℃之间。两种探针均仅与结核分支杆菌及BCG有杂交信号,而与其它受试分支杆菌及非分支杆菌杂交结果都呈阴性。它们的特异性都很强,但188bp探针的敏感性约是寡核苷酸探针的7～16倍,而且188bp探针检测本底较低,是检测结核分支杆菌的较佳选择     

An inexpensive and simple method for thermally stable immobilization of DNA on an unmodified glass surface: UV linking of poly(T)10-poly(C)10-tagged DNA probes

Microarrays printed on glass slides are often constructed by covalently linking modified oligonucleotide probes to a derivatized surface at considerable expense. In this article, we demonstrate that 14-base oligonucleotides with a poly(T)10 - poly(C)10 tail (TC tag), but otherwise unmodified, can be linked by UV light irradiation onto a plain, unmodified glass surface. Probes immobilized onto unmodified glass microscope slides performed similarly to probes bound to commercial amino-silane-coated slides and had comparable detection limits. The TC-tagged probes linked to unmodified glass did not show any significant decrease in hybridization performance after a 20 min incubation in water at 100 degrees C prior to rehybridization, indicating a covalent bond between the TC tag and unmodified glass. The probes were used in thermal minisequencing cycling reactions. Furthermore, the TC tag improved the hybridization performance of the immobilized probes on the amino-silane surface, indicating a general benefit of adding a TC tag to DNA probes. In conclusion, our results show that using TC-tagged DNA probes immobilized on an unmodified glass surface is a robust, heat-stable, very simple, and inexpensive method for manufacturing DNA microarrays.     

The messenger ribonucleic acid content of Bacillus subtilis 168

Bacillus subtilis 168 messenger RNA was determined by DNA-RNA hybridization techniques, with denatured DNA immobilized upon cellulose nitrate membrane filters. The following results were obtained. (1) Cultures of B. subtilis, growing exponentially in enriched glucose-salts medium at 37 degrees , incorporated [5-(3)H]uracil into both ribosomal and messenger RNA fractions without the kinetic delay expected from the presence of the intracellular nucleotide pools. (2) However short the time of labelling with exogenous labelled uracil (down to 7sec.), 32-36% of the rapidly labelled RNA was messenger RNA and 68-64% was an RNA with the hybridization characteristics of ribosomal RNA. Analysis of the apparent nucleotide base composition of total (32)P-labelled rapidly labelled RNA and the two RNA fractions separated by hybridization at a DNA/RNA ratio 5:1 confirmed this finding. Of the rapidly labelled RNA, 31% readily hybridized with DNA at low DNA/RNA ratios and had an apparent base composition like that of the DNA, whereas 69% was hybridized only at low efficiency at low DNA/RNA ratios and had a composition identical with that of ribosomal RNA. (3) In cultures dividing every 48min. at 37 degrees , kinetic analysis of RNA labelled over a 20min. period showed that the average life-time of messenger RNA was 2.7-3.0min. and that its amount was 3.0% of the total RNA. (4) The hybridization of (3)H-labelled randomly labelled RNA with DNA at a DNA/RNA ratio 5:1 showed that 2.9% of the randomly labelled RNA had the characteristics of messenger RNA. (5) Experiments carried out as described by Pigott & Midgley (1968) indicated that hybridization at low DNA/RNA ratios (5:1) effectively accounted for all the messenger RNA in a given specimen. The efficiency coefficient of RNA hybridization lay within the range of 90-95% input, if an excess of DNA sites was offered for RNA binding. (6) These measurements are compared with other results obtained by different methods, and reasons for any major disagreement are suggested.     

Single nucleotide polymorphism discrimination assisted by improved base stacking hybridization using oligonucleotide microarrays

Efficiencies of mismatch discrimination using size-varied capture probes were examined at various hybridization temperatures. The probes were 17, 15, 13, 11, 9, and 7 nucleotides long and contained single-base mismatches at their 3' ends. The optimal signal intensity and efficiency of base stacking hybridization on mismatch discrimination were observed for capture probes with a melting temperature (Tm) value of 36 degrees C, in the detection of DNA sequence variations at 40 degrees C. We employed asymmetric PCR to prepare single-stranded target DNA labeled with a fluorescent dye, and the PCR product was hybridized on the DNA microarray with no further purification. Our efforts have enhanced the sensitivity and simplified the procedures of base stacking hybridization on mismatch discrimination. As a model experiment, this improved technology was used to identify plasmid templates of human leukocyte antigen (HLA)-A alleles 2601, 2902, and 0206 on oligonucleotide microarrays. It is now possible to apply this simple, rapid, sensitive, and reliable base stacking hybridization technology to detect DNA sequence variations on microarrays in clinical diagnosis and other applications.     

Biotinylated probes to detect Leptospira interrogans on dot blot hybridization or by in situ hybridization

Total genomic biotinylated probes which can identify leptospires by hybridization on filters or by in situ hybridization are described in this study. According to the weak G + C content of the strains studied (35-39%) and owing to the decreasing melting temperature (Tm) due to overbiotinylation, hybridization and wash temperatures were optimized at 33 degrees C and at 42 degrees C respectively. Fourteen serovars of Leptospira interrogans belonging to 11 different serogroups and three serovars of Leptospira biflexa were used in this study. Cross-hybridization results show that it is possible, by means of such probes, specifically to recognize pathogenic strains. These probes did not hybridize with the three saprophytic strains: L. buenos-aires, L. patoc and L. andamana. We also ran a total genomic probe, specific to the serovar buenos-aires which hybridizes only with homologous DNA.     

Sensitive non-radioactive dot-blot hybridization using DNA probes labelled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence.

A new labelling method for cloned DNA probes used in hybridization assays is described. The DNA insert of recombinant plasmid DNA was made partially single-stranded for the labelling reaction by a restriction enzyme digest, followed by a controlled exonuclease III incubation. A thiol-containing psoralen derivative was covalently bound through irradiation with UV-light to the remaining double-stranded region of the plasmid DNA. The psoralen-SH groups were labelled with a large number of metal chelators (diethylentriamine pentaacetic acid, DTPA) using poly-L-lysine as a macromolecular carrier. The main advantage of the labelling procedure is that a high degree of labelling is achieved without modification of the single-stranded DNA hybridizing sequences. The specific hybrids were labelled after filter hybridization with europium ions through the chelating groups of DTPA. The europium ions were quantitatively detected by time-resolved fluorometry. The sensitivity of the assay for target DNA detection was in the low picogram range, comparable to radioactively labelled DNA probes.     

Detecting minimal traces of DNA using DNA covalently attached to superparamagnetic nanoparticles and direct PCR-ELISA

A single bond covalent immobilization of aminated DNA probes on magnetic particles suitable for selective molecular hybridization of traces of DNA samples has been developed. Commercial superparamagnetic nanoparticles containing amino groups were activated by coating with a hetero-functional polymer (aldehyde-aspartic-dextran). This new immobilization procedure provides many practical advantages: (a) DNA probes are immobilized far from the support surface preventing steric hindrances; (b) the surface of the nanoparticles cannot adsorb DNA ionically; (c) DNA probes are bound via a very strong covalent bond (a secondary amine) providing very stable immobilized probes (at 100 degrees C, or in 70% formamide, or 0.1N NaOH). Due to the extreme sensitivity of this purification procedure based on DNA hybridization, the detection of hybridized products could be coupled to a PCR-ELISA direct amplification of the DNA bond to the magnetic nanoparticles. As a model system, an aminated DNA probe specific for detecting Hepatitis C Virus cDNA was immobilized according to the optimised procedure described herein. Superparamagnetic nanoparticles containing the immobilized HCV probe were able to give a positive result after PCR-ELISA detection when hybridized with 1 mL of solution containing 10(-18) g/mL of HCV cDNA (two molecules of HCV cDNA). In addition, the detection of HCV cDNA was not impaired by the addition to the sample solution of 2.5 million-fold excess of non-complementary DNA. The experimental data supports the use of magnetic nanoparticles containing DNA probes immobilized by the procedure here described as a convenient and extremely sensitive procedure for purification/detection DNA/RNA from biological samples. The concentration/purification potential of the magnetic nanoparticles, its stability under a wide range of conditions, coupled to the possibility of using the particles directly in amplification by PCR greatly reinforces this methodology as a molecular diagnostic tool.     

Molecular characterization of the Spirometra mansonoides genome: renaturation kinetics, methylation, and hybridization to human cDNA probes

High molecular weight DNA from pleroceroid larvae of the tapeworm Spirometra mansonoides was purified from isolated nuclei by conventional techniques. The DNA so isolated has a melting temperature (Tm) of 87 degrees C and a guanine plus cytosine (G/C) content of 44%. 5-Methyl cytosine could not be detected in plerocercoid DNA by HPLC analysis of DNA hydrolysates, by radiolabeling 5'-termini of MspI digests with polynucleotide kinase, or by comparing restriction patterns generated by MspI and HpaII. Renaturation kinetics demonstrated that the genome of S. mansonoides contains repetitive as well as single copy sequences and has a genome size estimated at approx. 1.6 X 10(9) bp. Hybridization was carried out between plerocercoid DNA and cDNAs for human beta-actin, alpha-tubulin and growth hormone (hGH). Rationale for this analysis was based on known homologies among actin and tubulin genes in numerous species and on apparent similarities between hGH and a plerocercoid growth factor that may be reflected in similar DNA sequence. Scanning densitometry of dot blots demonstrated that the hGH probe annealed to the same extent at low stringency (1 M NaCl, 55 degrees C) to DNA from plerocercoids, rat liver and chicken erythrocytes; but this interaction was less than to DNA from human lymphocytes, calf thymus and mouse skin. Similar results were obtained when restriction endonuclease digests of these DNAs were analyzed by Southern transfer. Little or no hybridization of the growth hormone probe to plerocercoid DNA was evident at higher stringency (1 M NaCl, 65 degrees C). In contrast, human tubulin and actin probes showed extensive hybridization to pleroceroid restriction fragments under the high stringency conditions.     

Immobilisation of DNA to polymerised SU-8 photoresist

SU-8 is an epoxy-based photosensitive resist, which is currently used for a large variety of MEMS and lab-on-a-chip applications. Here, we demonstrate a one-step process to functionalize SU-8 with DNA probes. The immobilisation procedure relies on direct coupling of DNA to SU-8 and resulted in surfaces with functional capture probe densities of approximately 10 fmol/mm(2) as determined by hybridisation assays with fluorescent labelled target molecules. A comparable density of functional capture probes was measured on commercial aldehyde coated glass. DNA probes did not decrease in hybridisation performance after 10 min incubation in water at 98 degrees C prior to hybridisation, indicating a covalent bond between DNA and SU-8. Finally, DNA microarrays of high quality were obtained on SU-8 by contact printing of probe solution directly on SU-8 demonstrating a simple method for the implementation of microarrays in microsystems.     

Preparation of radioiodinated simian virus 40 DNA for use in DNA - DNA reassociation kinetics experiments.

A method is described for the preparation of 125I-labelled SV40 DNA. Using this method, SV40 DNA can be routinely labelled to 15 - 10(6) dpm per mug; much higher specific activities are easily obtained by minor modifications of the method. Once incorporated, the radioactive label dissociates from DNA exceedingly slowly at 4 degrees C or at 68 degrees C. Iodinated SV40 DNA is shown to be useful in the quantitation of viral nucleic acid sequences in SV40-transformed 3T3 cells by DNA - DNA reassociation kinetics.     

Effect of radiofrequency radiation on mRNA expression in cultured rodent cells

Four rodent cell lines were exposed to 2450 MHz microwave radiation at a Specific Absorption Rate (SAR) of 103.5 +/- 4.2 W/kg for varying lengths of time at 37 degrees, 40 degrees, 42 degrees and 45 degrees C. mRNA was extracted from microwave-exposed and sham-exposed cells and dot blotted or Northern blotted to nitrocellulose. Radioisotope labelled DNA probes of oncogenes, heat shock protein or long terminal repeat sequences were hybridized to the mRNA, and the resulting autoradiographs analyzed for differences in levels of mRNA expression between exposed and nonexposed samples. With the cell lines and probes used in this study no significant differences in mRNA expression were observed after microwave exposure.     

Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes

We present a study on the refinement of filter-hybridization conditions for a series of synthetic oligonucleotides in the range from 17 to 50 base residues in length. Experimental conditions for hybridization and the subsequent washing steps of the filter were optimized for different lengths of the synthetic oligonucleotides by varying the formamide concentration and washing conditions (temperature and monovalent cation concentration). Target DNA was immobilized to the nitrocellulose filter with the slot blot technique. The sequences of the synthetic oligonucleotides are derived from the third exon of the human oncogene c-myc and the corresponding viral gene v-myc and the G + C content was between 43 and 47%. Optimal conditions for hybridization with a 82% homologous 30-mer and 100% homologous 17-, 20-, 25-, 30-, and 50-mers were found to be a concentration of formamide of 15, 15, 30, 30, 40, and 50%, respectively. Optimal conditions for washing were 0.5X standard sodium citrate (SSC) at 42 degrees C for 2 X 15 min. The melting temperature for these optimal hybridization and washing conditions was calculated to be up to 11 degrees C below the hybridization temperature actually used. This confirms that the duplexes are more stable than expected. The melting points for 17-, 20-, and 30-mers were measured in the presence of 5X SSC and found to be 43, 58, and 60 degrees C, respectively. Competition between double- and single-stranded DNA probes to the target DNA was investigated. The single-stranded DNA probes were about 30- to 40-fold more sensitive than the double-stranded DNA probes.     

Use of the polymerase chain reaction and oligonucleotide probes for the rapid detection and identification of Carnobacterium species from meat.

The polymerase chain reaction (PCR) was used selectively to amplify specific rDNA sequences of Carnobacterium divergens, C. mobile, C. piscicola and C. gallinarum in purified DNA extracts, crude cell lysates and food samples. The PCR products were visualized by agarose gel electrophoresis and identified, at species level, by hybridization reactions with three specific oligonucleotide probes for C. divergens, C. mobile and C. piscicola/C. gallinarum designed from 16S rRNA sequence data. The PCR was sufficiently sensitive to amplify DNA from a single bacterium to detectable levels after 30 cycles of amplification. Both radioactive (32P) and non-radioactive alkaline phosphatase labelled probes was able to detect the PCR products. Detection was highly specific and the probes did not hybridize with DNA samples from any other of the bacterial species tested. These methods enabled the rapid and specific detection and identification of carnobacteria from pure cultures and samples of meat.     

A combined PRINS-FISH technique for simultaneous localisation of DNA sequences on plant chromosomes

A novel approach for simultaneous localization of two DNA sequences on plant chromosomes is described. The approach is based on a combined use of primed in situ DNA labelling (PRINS) with fluorescent in situ hybridization (FISH). Traditionally, this has been done using FISH with two probes labelled by two different marker molecules. Compared to this method, the combined PRINS-FISH procedure is faster. Furthermore, because one of the DNA sequences is localized by PRINS with specific primers, only one labelled probe is needed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

Allele-specific hybridization to a DNA microarray can be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose mutations in the human beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.     

A procedure for repeated usage of 33P-labeled DNA probes in hybridization experiments.

We describe a technique for repeated use of 33P-labeled DNA probes in Southern hybridization experiments. A nick-translated 33P-labeled DNA probe in a volume of 0.5-1.0 ml of hybridization mixture (final concentration, 10-100 ng/ml) is used to wet a sheet of filter paper (approx 10 microliters/cm2), which covers a nylon membrane with DNA transferred by Southern blotting, and both are set between two washed X-ray films. The "sandwich" is placed in a plastic bag for hybridization for 16-24 h at 42 degrees C. This very simple procedure using 33P-labeled DNA probes has a number of advantages over the standard method using 32P-labeled probes: (a) a significantly lower biohazard (body/arms exposure); (b) a very small volume of hybridization mixture in contact with a DNA-containing membrane and the higher probe concentrations attainable, causing some increase in sensitivity, and, finally, (c) repeated use of the probe-containing filter (over approx 3 days for unique sequences and up to 2 weeks for reiterated sequences) due to a relatively long 33P half-life (25.3 days).     

Sepcific fragmentation of DNA heteroduplex molecules of two bacteriophage lambda mutants with endonuclease Si from Aspergillus oryzae.

Heteroduplex DNA molecules of two bacteriophage mutants (lambda b2 and lambda i434ct68) were obtained by the method of molecular hybridization. These heteroduplexes possessed two types of loops formed as a result of: a) deletion in one of the DNA strands; and b) substitution of a DNA fragment for nonhomological one. The digestion of heteroduplexes with single-stranded specific nuclease SI from Aspergillus oryzae produced two fragments at 37 degrees C and three ones at 55 degrees C. The separation of fragments and determination of their molecular weight were carried out by means of electrophoresis in agarose. The molecular weights both measured and preliminarily calculated proved to be close. One of the fragments was identificated by its biological activity in CaCl2-dependent infectious system with helperphage.     

Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization

DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods. The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes. These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E. coli, contain two mismatches between the two heat-stable enterotoxins. Colonies were replicated from agar medium onto paper filters and lysed with alkali followed by steam; probes were end labeled. After overnight hybridization at 40 degrees C and washing at 50 degrees C, autoradiograms were exposed at -70 degrees C. Results were consistent with suckling-mouse tests for heat-stable enterotoxins. A stronger signal was obtained on paper filters than on nitrocellulose filters. Enterotoxigenic E. coli cells were detected when mixed with a 1,000-fold excess of nonenterotoxigenic E. coli cells. This procedure appears to be more acceptable for routine testing than the use of cloned DNA fragments, labeling by nick translation, and lysing colonies on nitrocellulose filters.