Double-stranded DNA Templates Research Articles

Reversible photopadlocking on double-stranded DNA

We describe a highly efficient method for reversible photocircularization of oligonucleotide (ODN) on a double-stranded DNA template: 5-carboxyvinyl-2'-deoxyuridine-containing ODN was reversibly circularized around the target sequence of the double-stranded plasmid DNA resulting in formation of a catenated plasmid.

The Human OCT-4 Isoforms Differ in Their Ability to Confer Self-renewal

OCT-4 transcription factors play an important role in maintaining the pluripotent state of embryonic stem cells and may prevent expression of genes activated during differentiation. Human OCT-4 isoform mRNAs encode proteins that have identical POU DNA binding domains and C-terminal domains but differ in their N-terminal domains. We report here the cloning and characterization of the human OCT-4B isoform. Human OCT-4B cDNA encodes a 265-amino acid protein with a predicted molecular mass of 30 kDa. Embryonic stem (ES) cell-based complementation assays using ZHBTc4 ES cells showed that unlike human OCT-4A, OCT-4B cannot sustain ES cell self-renewal. In addition, OCT-4B does not bind to a probe carrying the OCT-4 consensus binding sequence, and we demonstrate that two separate regions of its N-terminal domain are responsible for inhibiting DNA binding. We also demonstrate that OCT-4B is mainly localized to the cytoplasm. Overexpression of OCT-4B did not activate transcription from OCT-4-dependent promoters, although OCT-4A did as reported previously. Furthermore, transcriptional activation by human OCT-4A was not inhibited by co-expression of OCT-4B. Taken together, these data suggest that the DNA binding, transactivation, and abilities to confer self-renewal of the human OCT-4 isoforms differ.

A site-directed mutagenesis method utilising large double-stranded DNA templates for the simultaneous introduction of multiple changes and sequential multiple rounds of mutation: Application to the study of whole viral genomes

A new technique for conducting site-directed mutagenesis was developed. This method allows the colour selection of mutants through the simultaneous activation or deactivation of the α-peptide of β-galactosidase. Double-stranded DNA plasmids containing large inserts (at least 6.4 kbp in the present experiments) can be used as the mutational template. The method can efficiently create mutations at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. The utility of the method for the analysis of viral genomes was demonstrated by applying it to the mutagenesis of a full-length cDNA copy of RNA-1 of Cowpea mosaic virus (CPMV). Six single-site mutants were initially produced which gave a variety of phenotypes when inoculated on to plants. To confirm that the phenotypes were directly caused by the introduced mutations, a second round of mutagenesis was used to create revertants of two of the mutants. In both cases, the revertants had a wild-type phenotype, demonstrating that the original phenotype was, indeed, the result of the introduced mutation. Overall, the results show that the present technique is a powerful method for site-directed mutagenesis of large DNA fragments, such as whole viral genomes, for functional studies.

In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants withDpnI

In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with<i>DpnI</i>

Inhibition of Human Carcinoma and Neuroblastoma Cell Proliferation by Anti-c-myc siRNA

Suppression of c-myc proto-oncogene expression by small interfering RNA (siRNA) in human epidermoid carcinoma KB-3-1 and neuroblastoma SK-N-MC cell lines was investigated. The siRNA duplex targeted to the exon 3 of c-myc mRNA (siRNA-I) was prepared by in vitro transcription using T7 RNA polymerase and short double-stranded DNA (dsDNA) templates. siRNA-I was shown to efficiently decrease c-myc mRNA expression in both tumor cell lines and to arrest their proliferation. Incubation of KB-3-1 cells with 150 nM siRNA-I results in a 92% decrease in the c-myc mRNA level and an 83% decrease in the protein level. In SK-N-MC cells, 150 nM siRNA-I causes a 60% decrease in the c-myc mRNA level and a 55% decrease in the protein level. The reduction of the c-myc mRNA level correlates with the inhibition of cell proliferation; 150 nM siRNA-I causes a 2.5-fold reduction in the SK-N-MC proliferation rate and a 15-fold decrease in the proliferation rate and complete arrest of cell division in KB-3-1 cells. siRNA-I has little effect on proliferation of the IMR-32 cells that overexpress the N-myc but not the c-myc gene, demonstrating that siRNA-I antiproliferation activity is mediated by specific block of c-myc expression.

Comparison of Synthetic DNA Templates with Authentic cDNA Templates in Terms of Quantification by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction

Synthetic DNA templates were compared with authentic cDNA templates as standards for the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The single-stranded DNA template used here targeted the multidrug resistant transporter P-glycoprotein/MDR1. The double-stranded DNA template, targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was synthesized using an exonuclease-free large fragment E. coli DNA polymerase I. The human colon carcinoma cell line Caco-2 and human duodenum biopsies were used to prepare the authentic cDNA templates. The standard lines were comparable for the synthetic DNA templates and authentic cDNA templates. Long-term cryopreservation at -80 degrees C resulted in the destabilization of the synthetic single-stranded DNA template compared with the authentic cDNA templates in the case of MDR1, whereas for GAPDH, the stability of the synthetic double-stranded DNA template was comparable with that of the authentic cDNA templates. Even for the synthetic DNA templates, repetitive freeze-thawing resulted in destabilization, especially at lower concentrations, and degradation products might have interfered with the RT-PCR's efficiency. The synthetic DNA templates are better than the authentic cDNA templates, but more than 5 cycles of repetitive freeze-thawing should be avoided.

Simple purification of small RNAs from seeds and efficient detection of multiple microRNAs expressed in Arabidopsis thaliana and tomato (Lycopersicon esculentum) seeds

MicroRNAs (miRNAs) play critical roles in the development of animals and plants. Characterizing the stage- and tissue-specific expression of miRNAs that potentially regulate target transcription factor expression is becoming more important for understanding the regulatory mechanisms of critical events during plant development. A simple method for purifying small RNAs from seeds is described, as well as an efficient non-radioactive labelling system for making miRNA probes. In Arabidopsis thaliana seed extracts, low molecular-weight (LMW) RNAs (e.g. 5S rRNA, tRNA and miRNA) were separated from high molecular-weight (HMW) nucleic acids (e.g. 28S and 18S rRNA, mRNA and genomic DNA) by fractionation using isopropanol. HMW RNAs precipitated in 20% isopropanol, while most LMW RNAs remained in the supernatant. The purified LMW RNAs were used successfully for RNA gel blotting to detect miRNAs expressed in Arabidopsis and tomato (Lycopersicon esculentum) seeds. To increase the detection sensitivity of the microRNA probes, additional digoxigenin-labelled uridine triphosphates (UTPs) were incorporated into the miRNA probes by designing template oligo DNAs with three extra adenines (A) at each end of their sequence. These DNA oligomers were used to make double-stranded DNA templates for miRNA probe synthesis. This probe (termed AAAPLUS) exhibited stronger signals than normal probes. A technique was also developed to quickly screen expressed miRNAs in seeds using a miniblot system, which enabled simultaneous examination with multiple miRNA probes. This method provides a simple alternative to microRNA microarrays to identify the major miRNAs expressed in seeds.

In vitro Transcription and Purification of RNAs of Different Size

In vitro run-off transcription from a double-stranded DNA template by T7 RNA polymerase is an elegant way to obtain highly pure and uniform RNA oligonucleotides of lengths ranging from about 15 to several thousand nucleotides. Here we describe the different strategies applied and optimized in our laboratory to enzymatically synthesize RNAs as necessary when working at the interface of bioinorganic chemistry, coordination chemistry, RNA biochemistry and structural biology.

Straightforward Procedure for Internal Control of Real-Time Reverse Transcription Amplification Assays

Internal controls are crucial for reliable detection of pathogens in nucleic acid amplification assays. Without an internal control, a negative amplification result may mean that the sample contains no target or that the reaction failed. We recently described (1) the use of internal controls that consist of internal control oligonucleotides (ICOs) that contain little more than primer sites and a probe-binding site that contains mismatches to the actual target. ICOs can be obtained commercially and do not require a separate control-specific detection probe. Because of the mismatches, the control does not hybridize to the single detection probe during the fluorescence-acquisition step, and thus the probe binds and detects only nucleic acid derived from the pathogen. However, amplification of the ICO can be verified during melting analysis after PCR. Many important viral pathogens contain an RNA genome that must be transcribed to cDNA before amplification. To provide a control for both reverse transcription and amplification, an internal control must be an RNA molecule. The standard method for generating such a control involves directional cloning of a double-stranded DNA template into a plasmid vector carrying bacteriophage RNA polymerase promoter sequences. The RNA is then transcribed in vitro from the DNA template (2)(3). We have extended the ICO technique to RNA by introducing an RNA polymerase promoter and adding 2 simple preparation steps. We demonstrate the application of this control technique to the reverse transcription-PCR (RT-PCR) step of a LightCycler (Roche Molecular Biochemicals) real-time hybridization probe assay for respiratory syncytial virus (RSV). The sequences of the primers and probes for the RSV assay were 5′-GCCAAAAAATTGTTTCCACAATA-3′, 5′-TCTTCATCACCATACTTTTCTGTTA-3′, 5′-CY5.5-GGAATTCACATGGTCTACTACTGACTGT-phosphate, and 5′-GTTGTTCTATAAGCTGGTATTGATGCA-fluorescein-3′ (4). The 2-step LightCycler (Roche) RT-PCR was performed as follows. For the reverse transcription step (total volume of 10 μL), the reaction mixture consisted of 1.5 mM MgCl2, 2.5 …

Sequence-Selective Interaction of the Minor-Groove Interstrand Cross-Linking Agent SJG-136 with Naked and Cellular DNA: Footprinting and Enzyme Inhibition Studies

SJG-136 (3) is a novel pyrrolobenzodiazepine (PBD) dimer that is predicted from molecular models to bind in the minor groove of DNA and to form sequence-selective interstrand cross-links at 5'-Pu-GATC-Py-3' (Pu = purine; Py = pyrimidine) sites through covalent bonding between each PBD unit and guanines on opposing strands. Footprinting studies have confirmed that high-affinity adducts do form at 5'-G-GATC-C-3' sequences and that these can inhibit RNA polymerase in a sequence-selective manner. At higher concentrations of SJG-136, bands that migrate more slowly than one of the 5'-G-GATC-C-3' footprint sites show significantly reduced intensity, concomitant with the appearance of higher molecular weight material near the gel origin. This phenomenon is attributed to interstrand cross-linking at the 5'-G-GATC-C-3' site and is the first report of DNA footprinting being used to detect interstrand cross-linked adducts. The control dimer GD113 (4), of similar structure to SJG-136 but unable to cross-link DNA due to its C7/C7'-linkage rather than C8/C8'-linkage, neither produces footprints with the same DNA sequence nor blocks transcription at comparable concentrations. In addition to the two high-affinity 5'-G-GATC-C-3' footprints on the MS2 DNA sequence, other SJG-136 adducts of lower affinity are observed that can still block transcription but with lower efficiency. All these sites contain the 5'-GXXC-3' motif (where XX includes AG, TA, GC, CT, TT, GG, and TC) and represent less-favored cross-link sites. In time-course experiments, SJG-136 blocks transcription if incubated with a double-stranded DNA template before the transcription components are added; addition after transcription is initiated fails to elicit blockage. Single-strand ligation PCR studies on a sequence from the c-jun gene show that SJG-136 binds to 5'-GAAC-3'/5'-GTTC-3' (preferred) or 5'-GAGC-3'/5'-GCTC-3' sequences. Significantly, adducts are obtained at the same sequences following extraction of DNA from drug-treated K562 cells, confirming that the agent reaches the cellular genome and interacts with the DNA in a sequence-selective fashion. Finally, SJG-136 efficiently inhibits the action of restriction endonuclease BglII, which has a 5'-A-GATC-T-3' motif at its cleavage site.

Single-nucleotide-specific PNA-peptide ligation on synthetic and PCR DNA templates.

DNA-directed chemical synthesis has matured into a useful tool with applications such as fabrication of defined (nano)molecular architectures, evolution of amplifiable small-molecule libraries, and nucleic acid detection. Most commonly, chemical methods were used to join oligonucleotides under the control of a DNA or RNA template. The full potential of chemical ligation reactions can be uncovered when nonnatural oligonucleotide analogues that can provide new opportunities such as increased stability, DNA affinity, hybridization selectivity, and/or ease and accuracy of detection are employed. It is shown that peptide nucleic acid (PNA) conjugates, nonionic biostable DNA analogues, allowed the fashioning of highly chemoselective and sequence-selective peptide ligation methods. In particular, PNA-mediated native chemical ligations proceed with sequence selectivities and ligation rates that reach those of ligase-catalyzed oligodeoxynucleotide reactions. Usually, sequence-specific ligations can only be achieved by employing short-length probes, which show DNA affinities that are too low to allow stable binding to target segments in large, double-stranded DNA. It is demonstrated that the PNA-based ligation chemistry allowed the development of a homogeneous system in which rapid single-base mutation analyses can be performed even on double-stranded PCR DNA templates.

748. Generation of Novel Double-Stranded AAV Vectors with an Expanded Packaging Capacity Using Linearized 1-AAV ITR DNA Molecules

Introduction: The double-stranded AAV (dsAAV) vector is a new entity in the field of AAV based technologies. DsAAVs bypass a key requirement of AAV mediated transduction-conversion of the single stranded AAV genome into a double-stranded DNA template capable of RNA transcription. Currently, the only reported method for efficient and practical synthesis of double-stranded AAV's involves a standard AAV plasmid that has had one of its two AAV-inverted terminal repeat sequences mutated. Here we report a novel and practical method to synthesize double-stranded AAV vectors with a slightly increased packaging capacity as compared to the recently published technique.

The Antileukemia Drug 2-Chloro-2′-deoxyadenosine: An Intrinsic Transcriptional Antagonist

The nucleoside analog 2-chloro-2'-deoxyadenosine (CldAdo; cladribine) is effective in the treatment of hairy cell leukemia and chronic lymphocytic leukemia. CldAdo is phosphorylated and incorporated into cellular DNA but is not an absolute chain terminator. We demonstrated by in vitro gel-shift assays that binding interactions of the human TATA box-binding protein (TBP) were disrupted on 2-chlorodeoxyadenosine monophosphate (CldAMP)-substituted TATA box consensus sequences. We hypothesized that human RNA polymerase II (pol II) transcriptional processes would therefore be affected by 2-chlorodeoxyadenosine triphosphate (CldATP) incorporation into a promoter TATA element. Double-stranded DNA templates containing the adenovirus major late promoter and coding sequences were enzymatically synthesized as control or with site-specific CldAMP residues, incubated with HeLa extract, and the synthesis of radiolabeled 44-base transcripts was assessed. With increasing amounts of HeLa extract, CldAMP substitution for dAMP within the TATA box decreased in vitro pol II transcription by approximately 35% compared with control substrates. Time-course studies showed that transcript production increased in a linear fashion on control substrates. In contrast, transcription on CldAMP-substituted TATA sequences reached a plateau after 20 min. Furthermore, CldAMP-substituted promoter sequences trapped or sequestered TBP, preventing its dissociation from DNA and subsequent binding to additional TATA elements to reinitiate transcription. CldAdo thus represents the first example of a nucleoside analog that acts as a transcriptional antagonist. CldATP incorporation into gene regulatory sequences may provide a novel strategy to modulate specific protein/DNA interactions.

Elongation of oligonucleotide primers forming a triple helix on double-stranded DNA templates by purified DNA polymerases

Current knowledge on the replication of DNA involves enzymatic steps of DNA strand separation upon helicase activity, thus enabling the exposed bases of the single-stranded DNA to direct the polymerization of complementary nucleotides through Watson–Crick base pairing rules by DNA-dependent DNA polymerases. Here, we report that oligonucleotide primers (9–11 nt long) bound to the double-stranded DNA, can be elongated by the T7 and by the Thermus thermophilus DNA polymerases and by the Escherichia coli Klenow fragment. A perfectly base-paired DNA cannot be used as a template, but a single A/A mismatch located even 5 bp upstream from the theoretical 3 ′-end of the oligonucleotide primer is sufficient for DNA elongation by these first two enzymes, while five are required for the Klenow fragment. Elongation products from templates containing A/A mismatches at different positions revealed similar patterns independently of the positions of the A/A mismatches. The sequencing of the elongated products revealed that both purine and pyrimidine are incorporated at the pyrimidine–purine–pyrimidine transitions of the template strands, probably because of a shift of the primers on the double helix template. These data confirm that prokaryotic DNA polymerases may accommodate transiently three DNA strands in their catalytic centre. They also show for the first time that replication can occur on double-stranded DNA in the absence of DNA helicase, when mismatches are present in the vicinity of the triple helix initiation complex.

Preventing nondesired RNA-primed RNA extension catalyzed by T7 RNA polymerase.

The transcription patterns of 64 linear double stranded DNA templates obtained with T7 RNA polymerase were investigated. These templates consisted of 17 nucleotide-long sequences under the control of the minimal bacteriophage T7 promoter and represented all possible combinations of nucleotides at positions +8, +10 and +11. Two clearly distinct types of template were identified, which produced the range of transcription patterns observed: (a) those that yielded 17-nucleotide-long RNA as the only detectable run-off product (only 15% of the total), and (b) templates that in addition to the expected full-length RNA, produced other products longer than 17 nucleotides. Self-complementarity analysis of the expected run-off transcripts showed that those obtained from the first type of template were able to form stable intermolecular duplexes with non-base-paired 3'-ends. However, the second type of template yielded RNAs able to generate energetically favorable intermolecular duplexes with 3'-end complementarity, therefore yielding an RNA-primed RNA-template. The gel-purified 17-nucleotide-long RNAs transcribed from the latter yielded longer products when incubated under in vitro transcription conditions in the absence of a DNA template. No extension was observed when assaying the 17-nucleotide RNA products resulting from the first type of template. We observed that just a single nucleotide change within the DNA template could convert the RNA product from an RNA-primed template into a nonextendible dimer thus leading to a drastic switch of the 17-nucleotide product yield from less than 10% to 100%. Further, two type B DNA templates were extended by two nucleotides at the 3'-end, to produce RNA transcripts theoretically unable to form 3'-end base-paired duplexes. The full-length products of these modified DNA templates were found to be nonextendible by T7 RNA polymerase under the standard in vitro transcription conditions.

Pausing of DNA Polymerases on Duplex DNA Templates due to Ligand Binding in Vitro

Using the recently developed peptide nucleic acid (PNA)-assisted assay, which makes it possible to extend a primer on duplex DNA, we study the sequence-specific inhibition of the DNA polymerase movement along double-stranded DNA templates imposed by DNA-binding ligands. To this end, a plasmid vector has been prepared featuring the polylinker with two flanking priming sites to bi-directionally initiate the primer-extension reactions towards each other. Within this plasmid, we have cloned a set of random DNA sequences and analyzed the products of these reactions with several phage and bacterial DNA polymerases capable of strand-displacement synthesis. Two of them, ø29 and modified T7 (Sequenase 2.0) enzymes, were found to be most potent for primer extension in the presence of DNA-binding ligands. We used these enzymes for a detailed study of ligand-induced pausing effects with four ligands differing in modes of binding to the DNA double-helix. GC-specific intercalator actinomycin D and three minor groove-binders, chromomycin A 3 (GC-specific), distamycin A and netropsin (both AT-specific), have been chosen. In the presence of each ligand both selected DNA polymerases experienced multiple clear-cut pauses. Each ligand yielded its own characteristic pausing pattern for a particular DNA sequence. The majority of pausing sites could be located with a single-nucleotide resolution and corresponded to the preferred binding sites known from the literature for the ligands under study. Besides, DNA polymerases stalled exactly at the positions occupied by PNA oligomers that were employed to initiate the primer extension. These findings provide an important insight into the DNA polymerase performance. In addition, the high-resolution ligand-induced pausing patterns we obtained for the first time for DNA polymerase elongation on duplex DNA may become a valuable addition to the existing arsenal of methods used to monitor duplex DNA interactions with various DNA-binding ligands, including drugs.

Assay of Intrinsic Transcript Termination by E. Coli RNA Polymerase on Single-Stranded and Double-Stranded DNA Templates

This chapter describes the principal techniques used for biochemical characterization of transcript termination. The chapter focuses on intrinsic termination by Escherichia coli (E. coli) RNA polymerase on double-stranded (dsDNA) and single-stranded (ssDNA) DNA templates. These methods can be easily adapted to study transcript termination of other RNA polymerases and factor-dependent termination, or extended to study aspects of transcript elongation. To study intrinsic termination of E. coli RNA polymerase, 5ʹ biotinylated linear dsDNA templates generated by polymerase chain reaction (PCR) using one 5ʹ biotinylated oligonucleotide and one unmodified oligonucleotide. The template contains a promoter and an intrinsic termination site. Two solid-phase transcription methods are developed to prepare transcript elongation complexes halted at a distinct position along the DNA template. Use of biotinylated templates provides an attractive alternative to using T4 polynucleotide kinase and [γ 32 P] ATP to radiolabel one of two oligonucleotides required to amplify a linear DNA template by PCR.

Single-cell mRNA library analysis by Northern blot hybridization.

AbstractThe debut of RNA-polymerase cycling reaction (RNA-PCR) has promised to provide linear amplification of a reproducible mRNA library from as few as 20 single cells (1). By incorporating a RNA promoter element during the synthesis of double-stranded complementary DNA (cDNA) templates, a poly(A+) RNA library can be generated and reamplified from the templates in the same conformation and composition as its mRNA origins (Fig. 1). Using microarray analysis, the RNA-PCR-derived poly(A+) library has been proven to contain above 97% of the original poly(A+) RNA population and maintain 88±4% linear correlationship to the populationary ratio of each RNA species (Chapter 12). It has also been tested to generate a full-length mRNA library from as few as 20 homologous tissue cells (2-pg mRNAs) for profiling cancer stages in vivo. An illustration of using RNA-PCR-derived poly(A+) RNA for Northern blot analysis. The mRNA generated in step D can be fractionated on a formaldehydeagarose gel for specific gene detection. KeywordsLNCaP CellRadioactive ProbeHybridization ChamberKlenow EnzymemRNA LibraryThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A one-step method for in vitro production of tRNA transcripts.

Sequencing of a large number of microbial genomes has led to the discovery of new enzymes involved in tRNA biosynthesis and tRNA function. Preparation of a great variety of RNA molecules is, therefore, of major interest for biochemical characterization of these proteins. We describe a fast, cost-effective and efficient method for in vitro production of tRNA transcripts. T7 RNA polymerase requires a double-stranded DNA promoter in order to initiate transcription; however, elongation does not require a double-stranded DNA template. A partially double-stranded transcription template formed by annealing of a short oligonucleotide, complementary to the T7 promoter, to a larger oligonucleotide is shown to be a good substrate for in vitro transcription. This method allows rapid production of a variety of tRNA transcripts which can be aminoacylated well. This eliminates the need for cloning of tRNA genes, large-scale plasmid preparation and enzymatic digestion.

Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing™ technology

A new one-step method for fast and efficient preparation of double-stranded DNA template, suitable for use with Pyrosequencing™ technology, has been developed. In the new method, two different types of oligonucleotides were used to prevent reannealing of remaining PCR primers to the template: oligonucleotides complementary to the PCR primers and 3′-end modified oligonucleotides with the same sequence as the PCR primers. Advantages with the new strategy are: (i) faster and simpler template preparation procedure (one-step); (ii) no need for exonuclease I treatment; and (iii) less problem with unspecific priming from loop structures and dimers. By careful oligonucleotide design, and/or by addition of single-stranded DNA-binding protein, problems with unspecific sequence signals due to mispriming can be reduced. The new method was used for analysis of genotype variations within the renin–angiotensin–aldosterone system.