bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis of Oligoribonucleotides Using the 2‐Nitrobenzyloxymethyl Group for 2′‐Hydroxyl Protection
Authors: Miller Tod J. , Schwartz Miriam E. , Gough Geoffrey R. 
Published: June 2004

AbstractRNA can be rapidly synthesized using protected ribonucleoside phosphoramidite monomers that carry 2‐nitrobenzyloxymethyl groups on their 2‐hydroxyls. These protecting groups are removed from the oligoribonucleotide products by exposure to long‐wave UV light.
Synthesis and Purification of Oligonucleotide N3′→︀P5′ Phosphoramidates and their Phosphodiester and Phosphorothioate Chimeras
Authors: Fearon Karen L. , Nelson Jeffrey S. 
Published: June 2004

AbstractThis unit describes the synthesis and purification of oligonucleotide N3′→︀P5′ phosphoramidates, wherein each 3′‐oxygen is replaced by a 3′‐amine in the 2′‐deoxyribose ring. The synthesis of required monomers and application of the method to preparation of phosphodiester‐ and phosphorothioate‐containing chimera of phosphoramidate is also reported.
Locked Nucleic Acids: Synthesis and Characterization of LNA‐T Diol
Authors: Pfundheller Henrik M. , Lomholt Christian 
Published: June 2004

AbstractLocked nucleic acids (LNAs) are RNA derivatives that have an O‐methylene linkage between the 2 and 4 positions of the ribose. This leads to exceptionally high‐affinity binding to complementary sequences. LNAs are synthesized from a commercially available sugar, 1,2:5,6‐di‐O‐isopropylidene‐a‐D‐allofuranose. An efficient and simplified procedure is presented for synthesizing a glycol donor that can be used for synthesis of a variety of LNA monomers. Then, as an example, the synthesis of the thymidine analog of LNA from this glycol donor is presented. The protocols give high yields of the desired products and avoid the use of time‐consuming column chromatography.
Cellular Delivery of Locked Nucleic Acids (LNAs)
Authors: Braasch Dwaine A. , Corey David R. 
Published: June 2004

AbstractLocked nucleic acids (LNAs) are RNA derivatives that have an O‐methylene linkage between the 2 and 4 positions of the ribose. This leads to exceptionally high‐affinity binding to complementary sequences. They are synthesized using standard DNA/RNA synthesis methods, and have a negatively charged backbone that confers good solubility. This unit describes a method for the introduction of LNA oligomers into cells. A support protocol also describes the determination of melting temperatures for LNA oligomers.
Engineering Disulfide Cross‐Links in RNA Using Thiol‐Disulfide Interchange Chemistry
Authors: Cohen Scott B. , Cech Thomas R. 
Published: June 2004

AbstractProtocols for postsynthetic modification of 2‐amino‐containing oligoribonucleotides with either an alkyl‐phenyl disulfide or an alkyl thiol group are described. These groups react under mild conditions to form disulfide cross‐links by thiol‐disulfide interchange. These reactants do not form a disulfide bond when incorporated on opposite faces of a short continuous RNA helix, but do form disulfide bonds rapidly when they are placed in proximity. In addition, by incorporating these groups at various positions on large RNAs by semisynthesis, the dynamics of thermal motions can be detected. Such motions are believed to be linked to biological function, and the protocols presented in this unit are among the few simple ways to assess such dynamics.
NMR Determination of Oligonucleotide Structure
Authors: James Thomas L. 
Published: June 2004

AbstractThis unit provides an overview of the use of NMR to determine oligonucleotide structure. It covers basic NMR spectral properties, acquisition of interproton distance restraints and torsion angle restraints, structure refinement, assessment of the quality of the structure obtained. Software programs used in the process are also described.
RNA Secondary Structure Prediction
Authors: Mathews David H. , Turner Douglas H. , Zuker Michael 
Published: June 2004

AbstractDetails for predicting secondary structure of RNA sequences using free energy minimization are given. Protocols present the use of the computer program RNAstructure (for PCs) and the mfold server (for Unix platforms). The minimum free energy structure and a set of suboptimal structures with similar free energies are predicted. In addition, a triangular plot called the energy dot plot is predicted. This shows the superposition of all possible base pairs within a prescribed free energy increment.
Thermal Methods for the Analysis of RNA Folding Pathways
Authors: Draper David E. , Bukhman Yury V. , Gluick Thomas C. 
Published: June 2004

AbstractOnce a model of the secondary structure of an RNA has been deduced, thermal melting analysis can be used to determine whether the model accounts for all intramolecular interactions of the RNA, or whether noncanonical and tertiary interactions make the structure more stable than predicted, or link parts of the structure in unexpected ways. It is also useful to determine the pH, salt, and temperature ranges under which the RNA adopts a stably folded structure, or to analyze unfolding pathways. This unit discusses sample preparation, instrumentation, and theoretical background. It also provide a sample analysis of tRNA unfolding.
Use of Chemical Modification To Elucidate RNA Folding Pathways
Authors: Mathews David H. , Turner Douglas H. 
Published: June 2004

AbstractAs discussed in this overview, chemical modification is sensitive to the accessibility of a nucleotide to the solvent, and many nucleotides become less accessible as an RNA folds into its structured form. Chemical modification reagents are therefore suitable for following RNA folding, and can be used to study the kinetics of structure formation on time scales ranging from minutes to hours.
Key Experimental Approaches in DNA Nanotechnology
Authors: Seeman Nadrian C. 
Published: June 2004

AbstractDNA nanotechnology combines unusual DNA motifs with sticky‐ended cohesion to build polyhedral objects, topological targets, nanomechanical devices, and both crystalline and aperiodic arrays. The goal of DNA nanotechnology is control of the structure of macroscopic matter on the finest possible scale. Applications are expected to arise in the areas of X‐ray crystallography, nanoelectronics, nanorobotics, and DNA‐based computation. DNA and its close molecular relatives appear extremely well suited for these goals. This overview covers the generation of new DNA motifs, construction methods (synthesis, hybridization, phosphorylation, ligation), and a variety of methods for characterization of motifs, devices, and arrays. Finally, the use of DNA nanotechnology as a tool in biochemistry is discussed.
Chemoenzymatic Preparation of Nucleoside Triphosphates
Authors: Wu Weidong , Bergstrom Donald E. , Jo Davisson V. 
Published: June 2004

AbstractThe design and synthesis of alternative nucleoside triphosphate substrates for DNA and RNA polymerases holds continued promise to create biochemical probes and precursors for synthesis of nucleic acid mimics. The azole carboxamide nucleotides are of particular interest, as they display multiple conformations in the context of DNA replication. An efficient chemoenzymatic preparation of azole carboxamide deoxyribo‐ and ribonucleoside triphosphates is presented. Nucleoside diphosphate is prepared from nucleoside 5′‐O‐tosylate by displacement with tris(tetra‐n‐butylammonium) pyrophosphate. Enzymatic phosphorylation of the azole carboxamide deoxyribonucleoside diphosphate to its triphosphate is based on ATP as the phosphate donor and nucleoside diphosphate kinase as the catalyst, coupled with phospho(enol)pyruvate (PEP) and pyruvate kinase as an ATP regeneration system. Enzymatic phosphorylation of the azole carboxamide ribonucleoside diphosphate requires PEP as the phosphate donor and pyruvate kinase as the catalyst. The optimized purification uses boronate affinity gel to yield highly purified nucleoside triphosphate.
Introduction to the Synthesis and Purification of Oligonucleotides
Authors: Ellington Andrew , Pollard Jack D. 
Published: June 2004

AbstractModern nucleic acid synthesizers utilize phosphite triester chemistries that employ stable phosphoramidite monomers to build a growing polymer. These robust reactions allow easy generation of specific oligodeoxyribo‐ and oligoribonucleotides with a variety of labels, modified linkages, and nonstandard bases. Strategies are given for the maximization of synthetic yield, the generation of sequences containing site‐specific modifications, and the isolation of synthetic oligonucleotides. Protocols describe monitoring the progress of synthesis via the trityl assay and methods for deprotection.
Cerebral Infarct/Intracranial Cerebrovascular Disease
Authors: Grant P. Ellen , Schaefer Pamela W. , Gonzalez R. Gilberto 
Published: June 2004

AbstractImaging goals for intracranial cerebral vascular disease are (1) to assess the degree of parenchymal injury and identify intraparenchymal hemorrhage; (2) to determine if there are areas of altered perfusion that may be at risk for future injury; and (3) to assess the intracranial arteries (patency as well as direction of flow). This unit describes a that can be used to evaluate stable patients with acute, subacute, or chronic cerebrovascular symptoms. An is also given for cases of hyperacute strokes or cerebrovascular symptoms in an unstable patient.
Human Immunodeficiency Virus (HIV)
Authors: Auber Andrew E. , Belden Clifford 
Published: June 2004

AbstractMagnetic Resonance Imaging (MRI) for the evaluation of patients infected with human immunodeficiency virus (HIV), as with most other forms of intracranial inflammatory or infectious diseases, is a powerful though largely nonspecific diagnostic tool. For imaging of these complex patients with the varied and numerous pathologies they may harbor, the standard protocol is utilized to include gadolinium‐enhanced sequences. This unit presents optional imaging sequences, including magnetic resonance diffusion (dMRI), magnetic resonance perfusion (pMRI), and magnetic resonance spectroscopy (MRS), that can be employed should patient tolerance allow and if specific the clinical situation requires further clarification.
Aortic Dissection
Authors: Takahashi Naoki , Narra Vamsidhar 
Published: June 2004

AbstractThis unit presents a for imaging of thoracic aoprta to rule out aortic dissection. The core components of the imaging protocol are ECG (electrocardiograph)‐gated black blood HASTE (half Fourier single shot turbo spin echo), and contrast‐enhanced MR angiography (MRA). When no abnormal findings are seen in black blood HASTE or cine sequences, it is somewhat controversial whether contrast‐enhanced MRA is necessary. An based on a time of flight (TOF) sequence is presented which can be used instead of contrast‐enhanced MRA.
MRI of the Acute Injured Knee
Authors: Huegli Rolf W. , Tirman Phillip F.J. 
Published: June 2004

AbstractMagnetic Resonance Imaging (MRI) has become a mainstay in the assessment of internal derangement of the knee. MRI prior to surgery increases diagnostic confidence. It also influences clinical practice by identifying alternative diagnoses, e.g., for an osteochondral injury that clinically may mimic meniscal tears, different surgical approaches exist for many cases. This unit presents the standard protocol for imaging injured knees in a clinical setting. The parameters given in this unit are derived from a 1.5 T machine and may need to be altered slightly depending on the main magnetic field strength and the equipment manufacturer.
MRI of the Ankle and Hindfoot
Authors: Bencardino Jenny , Rosenberg Zehava S. 
Published: June 2004

AbstractMagnetic Resonance Imaging (MRI) is the diagnostic modality of choice for evaluation of traumatic ligamentous and tendinous injuries of the ankle and hindfoot. MRI is also valuable in the detection of occult bony trauma, osteochondral injuries, avascular necrosis, osteomyelitis, and a variety of other osseous conditions. This unit presents basic protocols for T1‐ and T2‐weighted sequences and short tau inversion recovery (STIR) imaging. Additional imaging following intravenous gadolinium is indicated in selected cases.
Magnetic Moment of a Spin, Its Equation of Motion, and Precession
Authors: Cheng Yu‐Chung Norman , Haacke E. Mark 
Published: June 2004

AbstractThis unit discusses the basic spin properties of the proton. The motion of a spin in an magnetic field that give rise to magnetic moment, is also discussed. The key to grasping much of MRI lies in understanding the concept of precession. Famous Bloch equation that basically leads to the description of the precession of a spin in the presence of an external magnetic field is also explained.
Screening Forms for Patients for MR Procedures and Individuals for the MR Environment
Authors: Shellock Frank G. 
Published: June 2004

AbstractThis appendix presents the screening forms that are to be filled by the patient prior to being scanned. Completion of these screening forms is also required for any person accompanying the patient into the magnet room. There are certain answers that will absolutely contraindicate the patient being scanned (e.g., if the patient has a cardiac pacemaker or ferromagnetic implant) while other questions are more designed to evaluate the patient's tolerance (such as whether they are claustrophobic). Other questions (such as those relating to renal disease) will determine if it is safe to use a contrast agent and make the physician aware of the medical status of the patient.
Digestion of DNA with Restriction Endonucleases
Authors: Bloch Kenneth D. , Grossmann Barbara 
Published: June 2004

AbstractRestriction endonucleases recognize short DNA sequences and cleave double‐stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. The first method presented in this unit is the cleavage of a single DNA sample with a single restriction endonuclease. A number of common applications of this technique are also described. These include digesting a given DNA sample with more than one endonuclease, digesting multiple DNA samples with the same endonuclease, and partially digesting DNA such that cleavage only occurs at a subset of the restriction sites. A protocol for methylating specific DNA sequences and protecting them from restriction endonuclease cleavage is also presented. A collection of tables describing restriction endonucleases and their properties (including information about recognition sequences, types of termini produced, buffer conditions, and conditions for thermal inactivation) is given at the end of the unit.
Mapping by Partial Endonuclease Digestions
Authors: Bloch Kenneth D. 
Published: June 2004

AbstractIn the procedure described in this unit, a DNA fragment radiolabeled at one of its two ends is purified by gel electrophoresis and subjected to partial cleavage by a restriction endonuclease. Analysis of the resulting products by polyacrylamide (or agarose) gel electrophoresis enables one to define the distance of restriction sites from the labeled end.
DNA‐Dependent DNA Polymerases
Authors: Tabor Stanley , Struhl Kevin , Scharf Stephen J. , Gelfand David H. 
Published: June 2004

AbstractThis unit presents characteristics and reaction conditions of the DNA‐dependent DNA polymerases, including E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, native and modified T7 DNA polymerase, and Taq DNA polymerase.
RNA Ligases
Authors: Tabor Stanley 
Published: June 2004

AbstractT4 RNA ligase, the product of the phage gene 63, is purified from phage‐infected cells. It catalyzes the ATP‐dependent covalent joining of single‐stranded 5'‐phosphoryl termini of DNA or RNA to single‐stranded 3'‐hydroxyl termini of DNA or RNA. This unit describes specific reaction conditions as well as applications such as radioactive labeling of 3' termini of RNA, circularizing deoxy‐ and ribo‐oligonucleotides, ligating oligomers for oligonucleotide synthesis, and stimulating the blunt‐end ligation activity of T4 DNA ligase.
Chemiluminescent Detection of Nonisotopic Probes
Authors: Perry‐O'Keefe Heather , Kissinger Carol M. 
Published: June 2004

AbstractWith recent advances in techniques for detecting chemiluminescent substrates, hybridization with nonisotopic rather than radiolabeled probes is becoming more common. In the , nylon membranes carrying transferred nucleic acids are prepared for hybridization with biotinylated probes by UV cross‐linking. This is a critical step in the procedure and the provides a detailed description of light‐source calibration. After hybridization, the target nucleic acid is detected through a series of steps that lead to an enzyme‐catalyzed light reaction. The describes chemiluminescent detection based upon antibody recognition of digoxigenin‐labeled probes. For both biotinylated and digoxigenin‐labeled probes, chemiluminescent detection is more sensitive than colorimetric detection and has the added advantage that the membrane can be used for multiple film exposures, then stripped and redetected with different probes.
Genomic DNA Libraries
Authors: Moore David D. 
Published: June 2004

AbstractGenomic DNA libraries are almost always screened by hybridization using a radioactive nucleic acid probe. Since this approach is essentially independent of a particular vector or type of target DNA, the main problem faced when considering creation of a genomic DNA library is simply generating a large enough number of recombinant DNA clones. The basic strategies used to address this problem have included both minimizing the number of clones necessary by incorporating large fragments of genomic DNA, and maximizing cloning efficiency by using vectors based on bacteriophage l. This unit discusses the appropriate numerical considerations for both ordinary genomic DNA libraries and subgenomic DNA libraries, and then describes a limited number of appropriate vectors.
Conversion of mRNA into Double‐Stranded cDNA
Authors: Klickstein Lloyd B. , Neve Rachael L. , Golemis Erica A. , Gyuris Jeno 
Published: June 2004

AbstractEnzymatic conversion of mRNA into double‐stranded insert DNA can be accomplished by a number of different procedures. All of them involve the action of reverse transcriptase and oligonucleotide‐primed synthesis of cDNA. After that, the procedures in common use diverge considerably. There are a number of methods for synthesizing the second strand and several procedures for producing suitable ends for making clonable DNA. The major goals of these procedures are to construct insert DNA that is as long as possible, with a high yield of conversion of mRNA into DNA that can ligate to vector DNA. The following protocols require only commercially available reagents and are usually successful in producing good cDNA libraries. The basic protocol describes a method for making blunt‐ended cDNA that can then be ligated to linkers for subsequent cloning into a unique restriction site such as EcoRI. The is a variation that requires fewer enzymatic manipulations and allows construction of directional cDNA libraries, which are particularly desirable when the goal is to generate expression cDNA libraries. The takes advantage of a linker‐primer consisting of (in order from 3' to 5') an oligo(dT) primer, a restriction site for the XhoI endonuclease, and a (GA)20 repeat to protect the restriction site during generation of the blunt‐ended cDNA. The internal XhoI sites on the individual cDNA molecules are protected by incorporation of 5‐methyl‐dCTP in the first‐strand nucleotide mix. The resulting cDNAs having unique ends can be cloned into EcoRI /XhoI ‐digested vectors after ligation of EcoRI adaptors to the 5' end and digestion by XhoI to release the 3' XhoI sites that were incorporated into the cDNA by the linker‐primer. These changes result in a considerably streamlined procedure that is substantially faster and easier than the basic protocol.
Using Synthetic Oligonucleotides as Probes
Authors: Duby Allan , Jacobs Kenneth A. , Celeste Anthony 
Published: June 2004

AbstractThe protocols in this unit describe procedures for using mixtures of
32P‐labeled oligonucleotides to screen recombinant DNA clones bound to nitrocellulose filters. A partial amino acid sequence of a protein is used to predict the nucleotide sequence of the gene that would encode it. A mixture of oligonucleotides is chosen that includes all possible nucleotide sequences encoding that amino acid sequence. This mixture of oligonucleotides is then used to screen a recombinant DNA library for the corresponding clones. In some cases however, the exact nucleotide sequence of a desired clone is known and it is possible to use a unique oligonucleotide as a probe.
Immunoscreening after Hybrid Selection and Translation
Authors: Velan Baruch 
Published: June 2004

AbstractIn this procedure, plasmid cDNA clones are screened for their ability to select a particular mRNA. Plasmid DNA is bound to nitrocellulose filters, hybridized to mRNA, washed, and the selected mRNA is eluted from the filter. Eluted mRNA is characterized by translation into
35S‐labeled protein, which is identified by immunoprecipitation and denaturing (SDS) polyacrylamide gel electrophoresis. The desired clone is that which is able to select an mRNA that translates into the desired protein. This procedure can be modified to characterize cosmids and bacteriophage DNA.
Overview of Strategies for Screening YAC Libraries and Analyzing YAC Clones
Authors: Chaplin David D. , Brownstein Bernard H. 
Published: June 2004

AbstractThis unit provides an introduction to the use of yeast artificial chromosome‐bearing yeast clones (hereafter referred to as YAC clones) in genome analysis. It describes criteria for designing a polymerase chain reaction (PCR) assay to be used in screening a YAC core library and discusses the rationale for verification and characterization of YAC clones obtained from these core laboratories. Protocols for maintaining YAC clones, analyzing YAC insert structure, preparing YAC DNA, and subcloning YAC inserts into other vectors are presented elsewhere in this volume.
Analysis of Isolated YAC Clones
Authors: Chaplin Divid D. , Brownstein Bernard H. 
Published: June 2004

AbstractThis unit provides a series of protocols describing the analysis and manipulation of an isolated YAC clone. The procedures are based upon the use of the YAC vector pYAC4. Once an isolated YAC clone has been obtained from a core laboratory, the clone can be analyzed as described herein. Methods for analysis involve growing and storing YAC‐containing yeast strains and purifying YAC DNA in a form suitable for assessing the size of the artificial chromosome and for conventional Southern blotting. Preparation of yeast chromosomes in agarose plugs for subsequent analysis by pulsed‐field gel electrophoresis is also described. Additional protocols are provided for recovering DNA fragments from the ends of a YAC genomic insert to be used as probes for detecting chimerism and for chromosome walking. Finally, preparation of high‐molecular‐weight YAC DNA is described and a general method for subcloning YAC inserts into cosmid or λ vectors for higher‐resolution analysis is provided.
Dideoxy DNA Sequencing with Chemiluminescent Detection
Authors: Martin Chris S. 
Published: June 2004

AbstractStandard dideoxy DNA sequencing can be performed easily and efficiently with nonisotopic, chemiluminescent detection by utilizing primers labeled with biotin in the sequencing reactions. As described in this unit, reaction products are separated by denaturing gel electrophoresis, transferred to a nylon membrane, and detected by first binding a streptavidin‐alkaline phosphatase conjugate, then incubating with a chemiluminescent 1,2‐dioxetane substrate. The emitted light signal is imaged on standard X‐ray film, producing high‐resolution DNA sequencing ladders. Indirect alkaline phosphatase‐labeling of biotinylated DNA with free streptavidin and biotinylated alkaline phosphatase is also detailed, Finally, the detection of sequencing reactions labeled with other haptens using specific antibody‐alkaline phosphatase conjugates is described.