Direct hybridization of labeled DNA to DNA in agarose gels

A naringinase assay capable of distinguishing between the content of naringin, prunin, and naringenin present in the incubation mixture, is described. The amount of these compounds can be estimated by combining two spectrophotometric procedures. (a) Treatment with strong alkali to determine the amount of nargingenin as well as the sum of naringin and prunin. (b) Assay of the liberated aldohexoses with o-aminodiphenyl. From the data thus obtained, the amount of the remaining substrate, the amount of the intermediate as well as the product at any given time can be calculated.     

Digoxigenin标记核酸探针分子杂交技术探讨

Dig标记和检测试剂盒中的封阻试剂配制成0.05%、0.1%、0.3%、0.5%、0.7%、0.9%六种浓度,65～68℃温度时,溶解时间分别为10、15、20、35、40、45分钟;预杂交,在免疫测定中进行封阻,背景反应最小,其次是不预杂交,在免疫测定中进行封阻,再次是预杂交,在免疫测定中不封阻;标记探针保存在-20℃,至少可稳定18个月,同一探针重复使用三次可获得满意效果.以上结果表明,DigDNA标记和检测系统将代替~(32)p标记及其检测系统.     

Compartmentation of guanine nucleotide precursors for DNA synthesis.

We have studied the kinetics of guanine incorporation into DNA in mouse T-lymphoma (S-49) mutant cells [PNPase (purine-nucleoside phosphorylase)- and HGPRTase (hypoxanthine: guanine phosphoribosyltransferase)-deficient] that are incapable of converting dGuo (deoxyguanosine) to Gua (guanine) ribonucleotides. Of the two possible pathways for an exogenous guanine source to reach DNA, firstly: dGuo----dGMP----dGDP----dGTP and secondly: Gua----GMP----GDP----dGDP----dGTP only the second pathway was found to be functional in providing guanine for DNA replication, although deoxyguanosine readily produced toxic cellular dGTP levels via the first pathway. The functional guanine-nucleotide-precursor pools for DNA are rather small; further, the depletion of the small GMP pool, but not that of GDP, GTP and dGTP, correlated well with the inhibition of DNA synthesis by mycophenolic acid, an IMP dehydrogenase inhibitor. These results support the hypothesis that guanine-nucleotide incorporation into DNA is highly compartmentalized and that a small functional guanine-nucleotide pool, e.g., the GMP pool, may serve a crucial role in limiting the availability of DNA precursor substrate.     

Comparison of different labeling methods for the production of labeled target DNA for microarray hybridization

Different labeling methods were studied to compare various approaches to the preparation of labeled target DNA for microarray experiments. The methods under investigation included a post-PCR labeling method using the Klenow fragment and a DecaLabel DNA labeling kit, the use of a Cy3-labeled forward primer in the PCR, generating either double-stranded or single-stranded PCR products, and the incorporation of Cy3-labeled dCTPs in the PCR. A microarray that had already been designed and used for the detection of microorganisms in compost was used in the study. PCR products from the organisms Burkholderia cepacia and Staphylococcus aureus were used in the comparison study, and the signals from the probes for these organisms analyzed. The highest signals were obtained when using the post-PCR labeling method, although with this method, more non-specific hybridizations were found. Single-stranded PCR products that had been labeled by the incorporation of a Cy3-labeled forward primer in the PCR were found to give the next highest signals upon hybridization for a majority of the tested probes, with less non-specific hybridizations. Hybridization with double-stranded PCR product labeled with a Cy3-labeled forward primer, or labeled by the incorporation of Cy3-labeled dCTPs resulted in acceptable signal to noise ratios for all probes except the UNIV 1389a and Burkholderia genus probes, both located toward the 3' end of the 16S rRNA gene. The comparison of the different DNA labeling methods revealed that labeling via the Cy3-forward primer approach is the most appropriate of the studied methods for the preparation of labeled target DNA for our purposes.     

Quantitative analysis of DNA hybridization in a flowthrough microarray for molecular testing

Quantitative information about the nucleic acids hybridization reaction on microarrays is fundamental to designing optimized assays for molecular diagnostics. This study presents the kinetic, equilibrium, and thermodynamic analyses of DNA hybridization in a microarray system designed for fast molecular testing of pathogenic bacteria. Our microarray setup uses a porous, nylon membrane for probe immobilization and flowthrough incubation. The Langmuir model was used to determine the reaction rate constants of hybridization with antisense targets specific to Staphylococcus epidermidis and Staphylococcus aureus strains. The kinetic analysis revealed a sequence-dependent reaction rate, with association rate constants on the order of 10⁵ M⁻¹ s⁻¹ and dissociation rate constants of 10⁻⁴ s⁻¹. We found that by increasing the probe surface density from 10¹¹ to 10¹² molecules/cm², the hybridization rate and efficiency are suppressed while the melting temperature of the DNA duplex increases. The maximum fraction of hybridized capture probes at equilibrium did not exceed 50% for hybridization with antisense sequences and was below 6% for hybridization with long targets obtained from PCR. The van’t Hoff analysis of the temperature denaturation data showed that the DNA hybridization in our porous, flowthrough microarray is thermodynamically less favorable than the hybridization of the same sequences in solution.     

Layer-by-Layer DNA film synthesis via branched hybridization

A new Layer-by-Layer (LbL) DNA film synthesis, where the driving force is the natural DNA hybridization, is presented in this work. The DNA films were synthesized via a branched mechanism and do not include any chemical binders between each layer as it is the case for film obtained by other synthesis paths. Kinetics of film formation were monitored by mass measurements with an electroacoustic network analyzer. The presented synthesis is a pathway to new DNA structures which can be used in biotechnologies and is complementary to those obtained by other LbL techniques.     

Enhanced photoelectrochemical method for linear DNA hybridization detection using Au-nanopaticle labeled DNA as probe onto titanium dioxide electrode

A photoelectrochemical method was proposed to detect DNA hybridization using Au nanoparticle modified DNA as one probe on TiO2 substrate, in which the TiO2 substrate was used not only as DNA anchors but also as the signal transducers. Hybridization between the probe and the target DNA oligonucleotides was confirmed by the decreased photocurrent of the TiO2 electrode. Compared with non-label probe, Au nanoparticles enhanced the photocurrent shifts after the hybridization. The photocurrent decreased with increasing the concentration of target DNA, indicating that this method could be used for quantitative measurements, and the discrimination of the complementary from mismatched DNA. Furthermore, the hybridization binding constant was obtained and photocurrent generation mechanism was discussed. The major advantages of this photochemical method are speed, simplicity and excellent specificity. This method provides a platform for studying a wide variety of biological processes using photoelectrochemical method.     

Chloroplast DNA synthesis in light and dark grown cultured Nicotiana tabacum cells as determined by molecular hybridization

A simple method using molecular hybridization was devised to quantitatively measure chloroplast DNA synthesis in vivo. Total cellular DNA isolated from Nicotiana tabacum suspension cells, labeled with ³H-thymidine, was hybridized to nitrocellulose membrane-bound cloned chloroplast DNA (ct DNA) fragments. Colorless, dark grown N. tabacum cells were found to contain approximately 3300–4800 chloroplast genome copies per cell, whereas light grown, green cells contain about 9500–12000 chloroplast genomes per cell. This difference in ct DNA levels suggests that the chloroplast genome is somewhat amplified during growth of the cells in the light. The hybridization technique was also used to measure the efficiency of hybridization between cloned spinach ct DNA and tobacco ct DNA. The two DNAs were found to cross-hybridize with an efficiency of 69–75%.     

Determination of hepatitis B virus DNA in serum by molecular hybridization

Hepatitis B virus (HBV) DNA was detected by direct spotting of alkali-denatured serum on a nitrocellulose filter and molecular hybridization with cloned HBV DNA as the probe. Measurement of the autoradiographic signals as the intensity of hybridization allowed the quantitation of HBV DNA content in serum specimens in reference to cloned HBV DNA. Direct spotting of denatured serum was approximately three times as sensitive as the conventional method in which proteinase-treated serum was extracted with phenol-chloroform. The intensity of hybridization with 25 specimens of HB virion concentrates correlated well with DNA polymerase activity (r = 0.89, P less than 0.01).     

Progress towards single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA

The enzymatic incorporation of modified dNTPs into a growing DNA strand has intensively been studied. Modifications were detectable reporter groups such as digoxigenin or biotin, fluorochromes or aliphatic side chains covalently attached to the base. Incorporation efficiencies were determined with several DNA polymerases using linear primer-extension reactions followed by denaturing PAGE as a high-resolution detection system. We describe the enzymatic synthesis of DNA consisting of modified nucleotides exclusively. A defined template-primer system allows us to trace incorporation: (1) in up to 18 neighboring positions for several dUTP-derivatives; or (2) in stretches of DNA of up to 40 bases in length with complete substitution of all four natural dNTPs by differently modified counterparts. Synthesized DNA molecules are shown to particularly exhibit dramatically altered physico-chemical properties by contrast with native DNA. These results provide a fundamental data set for probe generation in single-molecule DNA sequencing (SMS).     

Preparation of labeled nucleic acids (nick translation and iodination) for DNA homology and rRNA hybridization experiments

Nick-translated DNA preparations may contain very short fragments, causing inconsistent DNA homology results. This appears to be due to extensive fragmentation of the high molecular weight DNA used in the labeling procedure. DNA preparations from organisms containing DNA of low mol% G+C content appear to be the most fragmented. The problem was circumvented by labeling these DNA preparations with¹²⁵I. Sample preparation protocols have been developed for the routine¹²⁵I labeling of DNA and rRNA from a wide range of organisms.     

Simple chemical synthesis and hybridization properties of non-radioactive DNA probes

A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.     

Intra- and interpopulation differentiation of human genomes by molecular DNA hybridization

The DNA-DNA hybridization method was used to compare the repetitive sequences with a low degree of intragenomic divergence in various etno-territorial groups (Russians, Bouriats and Paleoasiats). Values of intergenomic divergence within groups and between them were estimated by a decrease in melting temperature of hybrid duplexes in relation to homologous 3H-labeled thermostable fraction reassociates of DNA of a Russian. Statistically valid differences in melting temperature were revealed when Russian, Bouriat and Paleoasiatic groups were compared. No such differences were found within each of the groups. Though the thermostability profiles had much in common in each case, some quantitative differences in melting temperature allowed to differentiate local groups in humans.     

Preparation of fluorescent-labeled DNA and its use as a probe in molecular hybridization

A method of the fluorescent-labeled DNA preparation for visualization of the complementary nucleotide sequences has been developed. Polynucleotide probes were alkylated randomly by 4-(N-methylamino-N-2-chloroethyl)-benzylamine followed by modification with such fluorochromes as dansyl chloride or fluorescein isothiocyanate (FITC). It was found that the FITC but not dansyl-labeled polynucleotides could serve as efficient probes when about 4% of nitrogen bases were modified. The conditions minimizing the loss of the alkylated bases from DNA were determined. The procedure for hybridization with FITC-labeled DNA as a probe is described, concentration of DNA probe being about 4 ng/mm2 of the nitrocellulose filter.