Detection of specific DNA sequences using antibodies recognizing UV-labelled DNA.

This non-isotopic method for detection of nucleic acids is based on the in situ labelling of the nucleic acid by exposure to UV-irradiation. The different UV-induced photoproducts, mainly of the thymidine dimer type, are recognized by purified rabbit antibodies specific to the lesions introduced. The UV-labelled nucleic acids can then be visualized by conventional immunostaining procedures. A major advantage of the technique is the low cost and the ease by which the DNA is specifically labelled. The purified rabbit antibodies were shown to be specific for UV-irradiated DNA, and the method was applied for detection of specific DNA sequences hybridized to homologous target DNA on membrane support. We believe that the sensitivity of the method can be improved, and the significance of using different UV-doses, immunostaining methods and membrane types is discussed.     

Okazaki pieces grow opposite to the replication fork direction during simian virus 40 DNA replication.

Simian virus 40 replicating DNA was pulse labeled with alpha-32P-dATP using an acellular DNA replication system. Nascent DNA chains of less than 200 nucleotides (Okazaki pieces) were then isolated from the denatured replicating DNA by electrosieving through a polyacrylamide gel column. The purified Okazaki pieces were hybridized to separated strands of Bg1(1)+Hpa1 simian virus 40 DNA restriction fragments immobilized on nitrocellulose filters. Only strands with polarity of the DNA replication fork direction hybridized with Okazaki pieces. Hence, Okazaki pieces in simian virus 40 are synthesized against the DNA replication fork direction.     

Opto-electronic DNA chip: high performance chip reading with an all-electric interface

Reading of DNA chips is usually based on fluorescence labeling of hybridised target molecules. Combined with the use of confocal fluorescence scanners, this approach shows very high performances in terms of accuracy and sensitivity. However, fluorescence readers remain costly and cumbersome. This prevents the use of DNA chips as a decentralised testing tool. Electrical monitoring of hybridisation is one way to reduce the cost and size of the reader. However, the multiplexing of electric detection-based systems in a miniaturised form remains challenging. Here, we present a system based on the use of a low cost CMOS photodetector array as a solid support for a DNA chip, coupled with revelation by enzyme-catalysed chemiluminescence. This system is shown to allow the detection of low pM target concentrations with a 3 logs dynamic range on dense DNA microarrays, with excellent inter-spot reproducibility. Combining electric interface and high analytical performances, this opto-electronic DNA chip is one attractive solution for nucleic acids detection and analysis in disposable, fully automatised, total analysis systems developed for decentralised testing.     

Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons

Nucleic acids ligation is a vital process in the repair, replication and recombination of nucleic acids. Traditionally, it is assayed by denatured gel electrophoresis and autoradiography, which are not sensitive, and are complex and discontinuous. Here we report a new approach for ligation monitoring using molecular beacon DNA probes. The molecular beacon, designed in such a way that its sequence is complementary with the product of the ligation process, is used to monitor the nucleic acid ligation in a homogeneous solution and in real-time. Our method is fast and simple. We are able to study nucleic acids ligation kinetics conveniently and to determine the activity of DNA ligase accurately. We have studied different factors that influence DNA ligation catalyzed by T4 DNA ligase. The major advantages of our method are its ultrasensitivity, excellent specificity, convenience and real-time monitoring in homogeneous solution. This method will be widely useful for studying nucleic acids ligation process and other nucleic acid interactions.     

Transferring DNA from electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose: properties of nucleosomes along mouse satellite DNA.

Electrophoresis fractionates nucleosomes which possess different protein compositions. We report here a procedure for transferring the DNA components of electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose (DBM-paper). Histones are first removed from nucleosome components by electrophoresis in the presence of cetyltrimethylammonium bromide (CTAB), leaving DNA fragments fixed within the original gel as the CTAB salts. The DNA is then converted to the sodium salt, denatured, and electrophoretically transferred to DBM-paper. The overall pattern of DNA on the resulting blot is visualized either by fluorography or by immunoautoradiography. This DNA pattern is then compared with autoradiograms obtained after hybridizing the same blot with specific 32P-labeled probes. Using mouse satellite DNA as a hybridization probe, we illustrate the above techniques and demonstrate that nucleosomes carrying satellite sequences are compositionally heterogeneous. The procedures described here should also be useful in the analysis of the nucleic acid components associated with other nucleoprotein complexes.     

The arrangement of simian virus 40 sequences in the DNA of transformed cells.

High molecular weight DNA, isolated from eleven cloned lines of rat cells independently transformed by SV40, was cleaved with various restriction endonucleases. The DNA was fractionated by electrophoresis through agarose gels, denatured in situ, transferred directly to sheets of nitrocellulose as described by Southern (1975), and hybridized to SV40 DNA labeled in vitro to high specific activity. The location of viral sequences among the fragments of transformed cell DNA was determined by autoradiography. The DNAs of seven of the cell lines contained viral sequences in fragments of many different sizes. The remaining four cell lines each contain a single insertion of viral DNA at a different chromosomal location. The junctions between viral and cellular sequences map at different places on the viral genome.     

Development of a novel DNA chip based on a bipolar semiconductor microchip system

We have applied an integrated circuit photodiode array (PDA) chip system to a DNA chip. The PDA chip system, constructed using conventional bipolar semiconductor technology, acts as a solid transducer surface as well as a two-dimensional photodetector. DNA hybridization was performed directly on the PDA chip. The target DNA, the Bacillus subtilis sspE gene, was amplified by polymerase chain reaction (PCR). The 340-bp PCR product was labeled using digoxigenin (DIG). A silicon nitride layer on the photodiode was treated with poly-L-lysine to immobilize the DNA on the surface of the photodiode detection elements. Consequently, the surface of the photodiode detector became positively charged. An anti-DIG-alkaline phosphatase conjugate was reacted with the hybridized DIG-labeled DNA. A color reaction was performed based on the enzymatic reaction between nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) staining solution and a DNA complex containing antibodies. A blue precipitate was formed on the surfaces of the photodiode detection elements. Successful quantitative analysis of the hybridized PCR products was achieved from the light absorption properties of the blue enzymatic reaction product that was produced after a series of reaction processes. Our DNA chip system avoids the complicated optical alignments and light-collecting optical components that are usually required for an optical DNA chip device. As a result, a simple, compact, portable and low-cost DNA chip is achieved. This system has great potential as an alternative system to the conventional DNA reader.     

The replication origin of proplastid DNA in cultured cells of tobacco

Summary When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.     

Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.     

A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.     

Renaturation of denatured DNA in NaI gradients

We show that denatured DNA from Tetrahymena mitochondria or phage T₇, partially renatures during centrifugation in NaI equilibrium density gradients. This makes these gradients unsuitable for the analysis of single-stranded complementary nucleic acids, especially if their complexity and mole percent G+C are low.     

Studies on the Mechanism of Replication of Adenovirus DNA II. The Nature of Single-Stranded DNA in Replicative Intermediates

Replicative intermediates of adenovirus type 5 DNA contain large stretches of single-stranded DNA. We have shown that this single-stranded DNA is mainly of parental origin, whereas all new DNA synthesized during one round of replication has a double-stranded structure. Hybridization experiments of the single-stranded DNA with isolated complementary strands of adenovirus type 5 DNA showed that this DNA hybridized only with the viral L-strand (the strand with the lower equilibrium density in alkaline CsCl) indicating that it represents the viral H-strand. This observation implies that replication always starts from one and the same molecular end. Electron microscopy of partially denatured Y-shaped intermediates confirmed this and showed that replication started from the molecular right end (the end richest in A-T base pairs). In conclusion, we have shown that replication of adenovirus type 5 DNA starts at the molecular right end, displacing the parental H-strand.     

A METHOD FOR the DETERMINATION of STRANDEDNESS of DNA FRAGMENTS CLONED IN PHAGE M13

Recombinant M13Hol phage containing Eco R1 restriction endonuclease fragments B, E, and F of adenovirus type 2 (Ad2) DNA were constructed by cloning into the unique Eco R1 site of the replicative form of the phage M13Hol176 DNA. Polarity of the adenovirus inserts in recombinant molecules was deduced by the following procedures: Viral DNA fragments obtained from Ad2 DNA molecules were purified, denatured, and subjected to electrophoresis. the separated DNA strands were transferred from agarose to nitrocellulose by the Southern procedure and hybridized with radioactive 3'-end labeled Hae III fragments of the recombinant phage DNAs. This procedure provided a rapid test for assaying strandedness of the cloned fragments.     

PNA/DNA interstrand cross-links from a modified PNA base upon photolysis or oxidative conditions

PNA/DNA interstrand cross-links (ICLs) were observed when peptide nucleic acids (PNAs) containing modified thymine derivatives were hybridized with the complementary or one-base mismatched DNA upon photolysis or treatments of oxidative agent. PNA/DNA ICL formation provides a useful method for biological applications such as antisense technologies or PNA chips.     

Reproducible and inexpensive probe preparation for oligonucleotide arrays

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.     

DNA modification of live cell surface

We report a novel approach for the attachment of DNA fragments to the surface of live cells. By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface. Method permits to control amount of immobilized DNA on the cell surface. All procedures can be completed within minutes and do not alter cell viability. Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion. Thus, this method allows rapid and simple DNA multicoding of the cell surface and, therefore, opens new opportunities in manipulating with cell–cell interactions.     

Identification of Yersinia enterocolitica using a random genomic DNA microarray chip

Aims: To fabricate a DNA chip containing random fragments of genomic DNA of Yersinia enterocolitica and to verify its diagnostic ability. Methods and Results: A DNA microarray chip was fabricated using randomly fragmented DNA of Y. enterocolitica. Chips were hybridized with genomic DNA extracted from other Y. enterocolitica strains, other Yersinia spp. and bacteria in different genera. Genomic DNA extracted from Y. enterocolitica showed a significantly higher hybridization rate compared with DNA of other Yersinia spp. or bacterial genera, thereby distinguishing it from other bacteria. Conclusions: A DNA chip containing randomly fragmented genomic DNA from Y. enterocolitica can detect Y. enterocolitica and clearly distinguish it from other Yersinia spp. and bacteria in different genera. Significance and Impact of the Study: A microarray chip containing randomly fragmented genomic DNA of Y. enterocolitica was fabricated without sequence information, and its diagnostic ability to identify Y. enterocolitica was verified.     

Fine mapping of secondary structures of fd phage DNA in the region of the replication origin.

A synthetic heptaribonucleotide, GACCCCC, which is complementary to a unique site on fd bacteriophage DNA, primes DNA synthesis of fd by T4 bacteriophage DNA polymerase. The rate of the GACCCCC-primed DNA synthesis was not uniform as reflected by the appearance of discrete DNA fragments as replication intermediates on an alkaline agarose gel. After 10 minutes of synthesis a significant fraction of the DNA product ran as a single band with a length of about 1960 nucleotides. We have isolated this DNA fragment, hybridized back to unlabeled fd DNA template, and mapped the Taq I restriction fragments by urea polyacrylamide gel electrophoresis. This fine mapping procedure has located two major pause sites at fd nucleotide positions 5575 and 5674. These sites reside in the stem of two very stable hairpin helices near the origin of DNA replication of fd. Models for the functional roles of these two hairpin helices are presented.     

Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA--streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates.

Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA-tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel-retardation assays.