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Results for “duplex sequencing”

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PubMed1991

Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA.

Eukaryotic 5S rRNA hybridizes specifically with 18S rRNA in vitro to form a stable intermolecular RNA:RNA hybrid. We have used 5S rRNA/18S rRNA fragment hybridization studies coupled with ribonuclease digestion and primer extension/chain termination analysis of 5S rRNA:18S rRNA hybrids to more completely map those mouse 5S rRNA and 18S rRNA sequences responsible for duplex formation. Fragment hybridization analysis has defined a 5'-terminal region of 5S rRNA (nucleotides 6-27) which base-pairs with two independent sequences in 18S rRNA designated Regions 1 (nucleotides 1157-1180) and 2 (nucleotides 1324-1339). Ribonuclease digestion of isolated 5S rRNA:18S rRNA hybrids with both single-strand- and double-strand-specific nucleases supports the involvement of this 5'-terminal 5S rRNA sequence in 18S rRNA hybridization. Primer extension/chain termination analysis of isolated 5S rRNA:18S rRNA hybrids confirms the base-pairing of 5S rRNA to the designated Regions 1 and 2 of 18S rRNA. Using these results, 5S rRNA:18S rRNA intermolecular hybrid structures are proposed. Comparative sequence analysis revealed the conservation of these hybrid structures in higher eukaryotes and the same but smaller core hybrid structures in lower eukaryotes and prokaryotes. This suggests that the 5S rRNA:16S/18S rRNA hybrids have been conserved in evolution for ribosome function.

Animals↗

PubMed2005

[Rapid analysis of genetically modified soybean by a duplex PCR-capillary electrophoresis system with laser-induced fluorescence detection].

OBJECTIVE: To develop a rapid detection method for genetically modified soybean resistant to glyphosate. METHODS: A duplex PCR was performed with primers designed in this study to simultaneously amplify heterogenous genes in transgenic soybean: CaMV-35S promoter, NOS terminator and CP4-EPSPS gene. And a simple capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed and applied to the rapid analysis of the above PCR products by using a 50 cm length x 100 microm i.d. capillary coated with linear polyacrylamide and an 8 g/L HPMC-4000 sieving buffer under 200 V/cm electric field strength. RESULTS: The proposed method was able to simultaneously detect the three heterogenous genes existing in genetically modified soybean under the optimization conditions of PCR and capillary electrophoresis. The measured sequences of the duplex PCR products were identical with the original genes' sequences. Moreover, the sample volumes required were not more than 5 nl and the detection could be completed in less than 24 min. The relative standard deviations (R. S. D.) of the migration times for the PCR products were < or = 3.2%. CONCLUSION: In comparison with agarose gels electrophoresis, the duplex PCR-based capillary electrophoretic method with laser-induced fluorescence detection is rapid, sensitive and accurate, and it is suitable for detection of genetically modified soybean.

DNA, Plant↗

PubMed1980

Structure and topology of 16S ribosomal RNA. An analysis of the pattern of psoralen crosslinking.

All of the eleven psoralen crosslinking features previously mapped by electron microscopy of the 16S rRNA can be matched by energetically stable duplex regions found by a computer search of the sequence. One additional particularly stable duplex found in the sequence has subsequently been seen in the microscope. There is no indication that a best or unique fit of the sequence to the crosslinking data has been found. However, it is encouraging that all twelve assignments involve non-overlapping regions of the sequence which allows their simultaneous construction. The resulting general pattern of secondary structure is very different from previous suggestions for rRNAs or other complex RNAs. It is an RNA chain folded in space much like a typical protein chain. Because of the interwound nature of double helices, base pairing between distant regions of the sequence might result in topologically knotted structures. However, examination of available electron microscopic data suggests that the 16S rRNA does not contain any knots.

Base Sequence↗

PubMed1993

Homologous DNA targeting with RecA protein-coated short DNA probes and electron microscope mapping on linear duplex molecules.

We demonstrate that RecA protein-coated, short single-stranded DNA probes paired with a specific homologous DNA sequence in a linear duplex target molecule and accurately targeted the selected DNA sequence. RecA protein-coated complementary ssDNA probes were reacted with linear duplexes, and the homologously paired molecules were observed by electron microscopy. The sites of interaction between the RecA protein-coated DNA probes and the uncoated duplex DNA targets were directly visible on individual target DNA molecules by high-resolution darkfield electron microscopy, without chemical fixation or sample shadowing. The efficiency and specificity of pairing were verified with 446 and 222 base single-stranded DNA probes that shared no homology with one another, and several linear duplex target DNAs with their respective probe homology sites at different locations with respect to the ends of the double-stranded DNA molecules. Measurements of the position of RecA protein-coated probes paired to individual target molecules, observed at high magnification, showed that DNA probes specifically paired at their corresponding homologous target sequences. This RecA protein-mediated DNA mapping method allows homologous sequence positioning and gene mapping on individual double-stranded DNA molecules. Targeting reactions in which two different probe/target sites were 900 bases apart on a single duplex target molecule allowed both sites to be mapped in the same targeting reaction; although targets displaying both probes simultaneously were seen much less frequently than expected. The possible torsional or mechanistic constraints related to these reactions are briefly discussed.

DNA Probes↗

PubMed1996

Solution structure of two mismatches G.G and I.I in the K-ras gene context by nuclear magnetic resonance and molecular dynamics.

Two mismatches, G.G and I.I, have been incorporated at the central position of 5'd-(GCCACXAGCTC).d(GAGCTXGTGGC) in order to carry out NMR and molecular dynamics studies. These duplexes constitute the sequence 29-39 of the K-ras gene coding for the glycine 12, a hot spot for mutation. The NMR spectra show that the duplexes are not greatly distorted by the introduction of the mismatches and their global conformation is that of a canonical B-form double helix. For the duplex containing the G.G mismatch, we propose for the major species, a type of pairing involving one hydrogen bond between the imino group of one central guanine and the carbonyl group of the opposite guanine. Both bases are in an anti conformation. Two conformations, with the same donor and acceptor pattern can coexist, one is obtained from the other by a 180 degrees rotation about the pseudodyadic axis. Exchange between the two forms is observed by NMR at low temperature. A minor species involving hydrogen bonding between the guanine amino group and the carbonyl group of the guanine on the opposite strand may also exist as shown by the molecular dynamics calculations. For the I.I mismatch we observe the same major species, i.e., hydrogen bonding between an imino proton of one base and the carbonyl group of the base on the opposite strand with both bases in an anti conformation. Exchange between these two conformations is faster than for the G.G mismatch. Further, we observe that the I.I mismatch adopts a minor conformation, in which one or other of the bases is in the syn conformation.

Chromosome Mapping↗

PubMed1998

A model for parallel triple helix formation by RecA: single-single association with a homologous duplex via the minor groove.

The nucleoproteic filaments of RecA polymerized on single stranded DNA are able to integrate double stranded DNA in a coaxial arrangement (with DNA stretched by a factor 1.5), to recognize homologous sequences in the duplex and to perform strand exchange between the single stranded and double stranded molecules. While experimental results favor the hypothesis of an invasion of the minor groove of the duplex by the single strand, parallel minor groove triple helices have never been isolated or even modeled, the minor groove offering little space for a third strand to interact. Based on an internal coordinate modeling study, we show here that such a structure is perfectly conceivable when the two interacting oligomers are stretched by a factor 1.5, in order to open the minor groove of the duplex. The model helix presents characteristics that coincide with known experimental data on unwinding, base pair inclination and inter-proton distances. Moreover, we show that extension and unwinding stabilize the triple helix. New patterns of triplet interaction via the minor groove are presented.

Computer Simulation↗

PubMed1980

Comparison of foldback sequences of herpes simplex virus types 1 and 2 DNA.

The DNAs of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) were separately denatured and allowed to renature briefly. The intrastand foldback structures that resulted from base pairing of inverted repeated sequences on otherwise single-stranded (ss) DNA were visualized in the electron microscope. The two genomes were found to contain similar size classes of small duplex stem DNA sequences. However, HSV-2 DNA appeared to possess an additional, larger size class of foldback structures not found on HSV-1 DNA. Both HSV DNAs were found to contain stem-plus-loop structures; the larger stem-plus-loop structures of the two genomes had similar stem lengths but dissimilar loop lengths. Thus, a comparison of the genomes of HSV-1 and HSV-2 showed that they possessed similar size classes of foldback sequences.

Base Sequence↗

PubMed2002

DNA binding properties of oligodeoxynucleotides containing pyrrolidino C-nucleosides.

We have incorporated pyrrolidino-C-nucleosides (pyrrolidino-pseudonucleosides) containing the base uracil and N-1-methyl uracil into oligodeoxynucleotides and compared their thermal duplex and triplex stabilities with unmodified or pseudouridine-containing oligodeoxynucleotides. We find relative destabilizations of triplex formation by ca. -13 to -1 degrees C per modification (relative to thymidine) in a strongly sequence dependent mode. Duplex formation is less destabilizing and more homogeneous with -4 to -2 degrees C per modification.

Base Sequence↗

PubMed2003

Stimulation of D-loop formation by polypurine/polypyrimidine sequences.

Most of the approaches used to correct gene mutations in mammalian cells involve the targeting of short nucleotide molecules to homologous chromosomal sequences and the replacement of resident sequences via homologous recombination and mismatch repair. The limited efficiency and inconsistent reproducibility of these techniques are major constraints to their use in gene therapy. One of the main problems is that it is impossible to obtain reproducible results when the targeted gene loci differ. We investigated the effects of flanking sequences on homologous recombination by means of an in vitro assay of the efficiency of oligonucleotide targeting to its homologous sequence on a large duplex molecule in a reaction catalysed by the Escherichia coli RecA protein. We demonstrated that polypurine.polypyrimidine tracts (PPTs) in duplex DNA strongly stimulate the formation of D-loops with short oligodeoxynucleotides. This result was reproduced with various PPT sequences and oligonucleotides. The stimulatory effect was observed at loci as far as 4000 bp from the PPT. The formation of complexes between the oligonucleotide and the duplex molecule depended on the extent of sequence similarity between the two DNAs and the presence of the RecA protein. The stimulatory effect was inhibited by excess RecA and restored by adding heterologous DNA. We suggest that PPT sequences induce conformational changes in duplex DNA, leading to the aggregation of molecules, facilitating homology searches. We compared, in vivo, the efficiency of the oligonucleotide-mediated correction of a URA3 chromosomal mutation for sequences with and without a PPT sequence in the vicinity. Consistent with our in vitro results, the efficiency of correction was eight times higher in the presence of the PPT sequence.

Base Sequence↗

PubMedDate not supplied

Synthesis of a cyclopentane amide DNA analogue and its base pairing properties.

cpa-DNA monomers containing the bases adenine and thymine have been synthesized starting from the known compound 1 in 12 steps. Partially and fully modified cpa-thymidine and cpa-adenosine containing oligodeoxynucleotides were synthesized by standard oligonucleotide chemistry. Fully modified homo-cpa-A sequences lead to duplex destabilization by -1.4 degrees C/mod. relative to DNA. As its congener bca-DNA, cpa-DNA prefers left-handed duplex formation where possible.

Adenine↗

PubMed1991

Selective inhibition of sequence-specific protein-DNA interactions by incorporation of 6-thioguanine: cleavage by restriction endonucleases.

Incorporation of the antileukemic agent 6-thioguanine (TG) into cellular DNA has been demonstrated to be a major determinant of its cytotoxicity. We have previously shown that complete replacement of G by TG within one DNA strand of the SV40 origin of replication can completely inhibit sequence-specific binding of the viral replication protein T antigen. The aim of the present study was to determine the effect of more selective TG substitutions on DNA-protein interactions, by utilizing the simpler base recognition sequence motifs of restriction endonucleases. In the first part of our study, we replaced G with TG in one or two of four possible sites within the duplex hexameric recognition sequence of BamHI (5'-G decreases GATCC-3'), by enzymatic extension of primed oligonucleotides. This extension was stalled, but not completely inhibited, at locations where insertion of consecutive TG moieties was required. Both strands of molecules containing a single substitution were cleaved by BamHI at reduced rates, with the substituted strand inhibited to a greater degree. In molecules containing two substitutions, neither strand was cut by BamHI. In contrast, we found that scission of these same mono- and disubstituted substrates by the less stringent isoschizomer MboI (5'-N decreases GATCN-3') was inhibited only slightly. In the second part of our study, we investigated the effect of analog substitution on scission by the type II-S enzymes AlwI and FokI, in order to separately determine the effects of restriction site modification versus scission site modification. We found that the reactivity of these enzymes was completely abolished by TG substitution within the recognition site, whereas substitution at the scission site had no effect. Our results demonstrate that infrequent TG substitutions within symmetric DNA sequences can inhibit sequence-specific interactions in an asymmetric fashion. In addition, although previous reports have shown that TG forms a relatively weak base pair with cytosine, it appears that the inhibition of restriction endonuclease-mediated cleavage resulting from TG incorporation is a function of the sequence requirements of the protein and not a general consequence of disrupted base-pairing at the recognition locus. These data support the idea that the cytotoxic consequences of TG incorporation may be due to inhibition of sequence-specific protein-DNA interactions.

Base Sequence↗

PubMed2006

Evaluation of elastic properties of atomistic DNA models.

A number of intriguing aspects in dynamics of double-helical DNA is related to the coupling between its macroscopic and microscopic states. A link between the elastic properties of long DNA chains and their atom-level dynamics can be established by comparing the worm-like chain model of polymer DNA with the conformational ensembles produced by molecular dynamics simulations. This problem is complicated by the complexity of the DNA structure, the small size of DNA fragments, and relatively short trajectory durations accessible in computer simulations of microscopic DNA dynamics. A careful study of all these aspects has been performed by using longer DNA fragments and increased durations of MD trajectories as compared to earlier such investigations. Special attention is paid to the necessary conditions and criteria of time convergence, and the possibility to increase the sampling by using constrained DNA models and simplified simulation conditions. It is found that dynamics of 25-mer duplexes with regular sequences agrees well with the worm-like chain theory and that accurate evaluation of DNA elastic parameters requires at least two turns of the double helix and approximately 20-ns duration of trajectories. Bond length and bond-angle constraints affect the estimates within numerical errors. In contrast, simplified treatment of solvation can strongly change the observed elastic parameters of DNA. The elastic parameters evaluated for AT- and GC-alternating duplexes reasonably agree with experimental data and suggest that, in different basepair sequences, the torsional and stretching elasticities vary stronger than the bending stiffness.

Base Pairing↗

PubMed1986

Archaebacteria: transcription and processing of ribosomal RNA sequences in Halobacterium cutirubrum.

The chromosome of Halobacterium cutirubrum contains a single ribosomal RNA gene cluster. The 5' to 3' organization of genes within this 6-kpb region is: 16S, alanine tRNA, 23S, 5S, cysteine tRNA. The entire gene cluster is transcribed as a single long primary transcript; processing of mature RNA sequences from the 5' region of the transcript begins prior to the completion of synthesis at the 3' end. There are five conserved octanucleotide direct repeats (TGCGAACG) in the 900-bp 5'-flanking sequence in front of the 16S gene. The positions of these repeat sequences correspond to the different 5' ends of the primary transcript and probably represent the RNA polymerase start sites. The 16S and 23S rRNA genes are surrounded by long nearly perfect inverted repeat sequences. These sequences probably form duplex structures in the primary transcript and are recognized by an RNaseIII-like endonuclease activity that carries out the initial excision of the precursor 16S and 23S rRNA sequences. These precursors are rapidly trimmed tot he mature 16S and 23S molecules and assembled into ribosomal particles. The processing sites for 5S rRNA appear to be at or very near to the mature ends of the 5S molecule. The tRNA sequences are processed with reduced efficiency from the primary transcript. Nuclease cuts have been detected at the ends as well as in the middle of the cysteine tRNA sequence suggesting that there may be alternative processing pathways, one resulting in proper excision of the mature tRNA sequence and the other resulting in improper excision and degradation of the tRNA sequence. The transcription termination sequence is believed to be at or beyond an AT-rich sequence preceded by a GC-rich sequence located distal to the cysteine tRNA gene.

Base Sequence↗

PubMed1995

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.

Antiviral Agents↗

PubMed1980

DNA sequence organization in the starfish Dermasterias imbricata.

The sequence arrangement in the genomic DNA of the starfish Dermasterias imbricata has been examined. Analyses of kinetics of reassociation in solution at DNA lengths of 400, 3100, and 6200 base pairs (bp) demonstrate the interspersion of repetitive and unique DNA. At a fragment length of 400 bp, 45% of the DNA reacts at a rate appropriate for single-copy sequences in a genome of this size (0.54 pg). Interspersion of repetitive sequences is also demonstrated by the reduced hyperchromicity of 3100- or 6200-bp fragments reacted to Cot 10, where only repetitive sequences have formed duplex. S1 nuclease digestion of 3100-bp fragments reassociated to Cot 10 shows that both short (approximately 230 bp) and long (greater than or equal to 2600 bp) repetitive sequences are present in this DNA. These data demonstrate a short period interspersion pattern in Dermasterias.

Animals↗

PubMed1980

The effects of base sequence and dangling bases on the stability of short ribonucleic acid duplexes.

Variable temperature proton nuclear magnetic resonance spectroscopy is used to establish that both sequence and dangling bases affect stability of short RNA double helices. Tetraribonucleotides, CAUG, UGCA and AGCU form reference duplexes (Tm 25 degrees, 33 degrees, 34 degrees, respectively) which contain an equal number of G . C and A . U base pairs and which show primary sequence is important. Pentaribonucleotides GAUGA, ACAUG, UGCAA and AGCUA, form duplexes (Tm 36 degrees, 35 degrees, 46 degrees, 45 degrees, respectively) with dangling adenines. Average Tm difference from the reference is +11 degrees for two 3' or 5'-dangling adenines. Pentaribonucleotides, CAUGU and UCAUG, form duplexes (Tm both 30 degrees) with dangling uracils. Average Tm difference from the reference is +5 degrees for two 3' or 5'-dangling uracils. Fraying was detected only in duplexes from the AGCU series. Presence of a dangling adenine, in duplex, AGCUA, caused a reduction in fraying.

Base Composition↗

PubMed1988

1H and 31P NMR investigations of actinomycin D binding selectivity with oligodeoxyribonucleotides containing multiple adjacent d(GC) sites.

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with adjacent 5'-GC-3' sites. ActD showed very high specificity for binding to GC sites regardless of oligomer length and surrounding sequence. For a first class of duplexes with a central GCGC sequence, a mixture of 1:1 complexes was observed due to the two different orientations of the ActD phenoxazone ring system. Analysis of 1H chemical shifts suggested that the favored 1:1 complex had the benzenoid side of the phenoxazone ring over the G base in the central base pair of the GCGC sequence. This is the first case in which an unsymmetrical intercalator has been shown to bind to DNA in both possible orientations. A unique 2:1 complex, with significantly different 1H and 31P chemical shifts relative to those of the 1:1 complexes, was formed with these same oligomers, again with the benzenoid side of the ActD molecule over the G base of the central GC base pair. There is considerable anticooperativity to binding of the second ActD in a GCGC sequence. In titrations of oligomers with the GCGC sequence, only the two 1:1 complexes are found up to ratios of one ActD per oligomer. Increasing the ActD concentration, however, resulted in stoichiometric formation of the unique 2:1 adduct. Spectrophotometric binding studies indicated that the apparent binding equilibrium constant for a GC site adjacent to a bound site is reduced by approximately a factor of 20 relative to the ActD binding constant to an isolated GC site.

Base Sequence↗

PubMed2005

Diagnosis of HNF-1alpha mutations on a PNA zip-code microarray by single base extension.

In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code microarray for the detection of hepatocyte nuclear factor-1alpha (HNF-1alpha) mutations that cause type 3 maturity onset diabetes of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 3' complementarity to the specific mutation site and 5' complementarity to the respective PNA zip-code sequence on the microarray. The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation sites in exon 2 of HNF-1alpha with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few successful applications of PNA in DNA chip technology.

DNA Mutational Analysis↗