bluuebunny/crossref_metadata_2025 huggingface dataset:
Rodent Models of Depression: Forced Swimming and Tail Suspension Behavioral Despair Tests in Rats and Mice
Authors: Porsolt Roger D. , Brossard Geneviève , Hautbois Carine , Roux Sylvain
Published: June 2004

Rodents forced to swim in a narrow space from which there is no escape will, after an initial period of vigorous activity, adopt a characteristic immobile posture, making only those movements necessary to keep their heads above the water. It was hypothesized that immobility reflected the animals' having learned that escape was impossible and their having given up hope. Immobility was therefore given the name “behavioral despair”. Immobility was subsequently found to be reduced by a wide range of clinically active antidepressant drugs. This simple behavioral procedure has since become a useful test for screening novel antidepressants in rats and is presented in this unit. An equivalent procedure in the mouse is also described along with a “dry” version of the test where immobility is induced simply by suspending the mouse by the tail.
Fear‐Potentiated Startle in Rats
Authors: Davis Michael
Published: June 2004

Fear‐potentiated startle measures the increase in the startle reflex elicited by a sudden noise in the presence of a cue that has previously been paired with footshock. It involves an initial training session in which a cue, such as a light, is paired with footshock, and then a later test session in which startle is elicited in the presence or absence of the cue. It thus involves associative learning, memory for that association measured during the retention test, and conditioned fear in the presence of the cue. Drugs that interfere with learning, memory, or fear and anxiety can alter fear‐potentiated startle depending on when they are given with respect to training and testing.
Latent Inhibition
Authors: Weiner Ina
Published: June 2004

Latent inhibition (LI) refers to the deleterious effects of nonreinforced stimulus preexposure on its subsequent conditioning. LI is considered to index an organism's ability to ignore inconsequential stimuli and as such provides a useful tool to measure attentional processes as well as their disruption and enhancement by various pharmacological, physiological and behavioral manipulations. During the last decade, LI has received increasing interest as an animal model of schizophrenia and of antipsychotic drug action. This unit describes the measurement of LI in the conditioned emotional response and two‐way active avoidance procedures, including parametric variations which allow the demonstration of the disruption and the potentiation of this phenomenon.
Ultrasonic Vocalizations in Rat and Mouse Pups
Authors: Hofer Myron A. , Shair Harry N. , Brunelli Susan A.
Published: June 2004

The ultrasonic vocalization (USV), or isolation calling response, of infant rats and mice has been studied as a measure of the intensity of an aversive affective state and as an early communicative behavior between pup and mother. The four protocols described in this unit are for the basic isolation testing procedure, and for elicitation of the contact quieting response to littermates and/or dam, the potentiation of isolation calling response by a prior brief maternal interaction and the predator‐induced suppression of USV by the scent of an unfamiliar male. These procedures for the elicitation of USV, and for its regulation by different kinds of social interaction, provides the basis for experimental research on the early development of emotion and communication in an animal model system.
Experimental Autoimmune Encephalomyelitis (EAE)
Authors: Racke Michael K.
Published: June 2004

The procedures described in this unit utilize murine models exclusively since murine EAE often results in a relapsing/remitting disease, similar to the early phase of most MS patients. EAE in the Lewis rat is a monophasic illness in which animals experience a single episode of paralysis from which most recover completely. This unit presents two methods for inducing EAE in mice: active induction and adoptive transfer
Animal Models of Retroviral Encephalopathies: Feline Model
Authors: Podell Michael , Buck Wayne R. , Hayes Kathleen A. , Gavrilin Mikhail A. , Mathes Lawrence E.
Published: June 2004

Human immunodeficiency virus infection in children and adults results in a progressive neurodegenerative disease consistent with a predominant subcortical mediated dementia. Techniques for developing a feline model of the early stages of lentiviral‐associated neurodegeneration are presented. The behavioral, neurophysiologic, immunologic, virologic, and neuropathologic aspects of this model are also described.
Inducing Photochemical Cortical Lesions in Rat Brain
Authors: Bergeron Marcelle
Published: June 2004

In the rat photochemical cortical lesion model described in this unit, an intravascular photochemical reaction induces endothelial damage resulting in platelet aggregation, thrombosis, thrombotic response (secretion of factors by the platelets) and permanent cerebral vascular occlusion. Because thrombosis is produced in pial vessels, the resulting cortical infarct is generally smaller and more reproducible than in the models involving occlusion of the middle cerebral artery. The surgical procedures involved are limited, making this model generally easier to perform and less invasive than most other models of permanent focal ischemia that involve mechanical occlusion of major cerebral arteries.
Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
Authors: Hawley‐Nelson Pamela , Ciccarone Valentina
Published: June 2004

The development of high‐efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipid‐based transfection reagents has accelerated biology research in studies of gene expression, control of cell growth, and cell lineage. In this unit, DNA transfection is described for adherent mammalian cells (both cells and primary cultures) along with an alternate procedure for enhanced transfection. A protocol is also described for transfection of suspension cells (lymphoid, myeloid and leukemic‐derived cells). In addition, transfection of RNA into adherent mammalian cells and DNA transfection into insect cells are presented. Importantly, a detailed procedure is described for optimization of reagents and transfection conditions.
Preparation of Bacterial Plasmid DNA
Authors: Engebrecht JoAnne , Heilig J.S. , Brent Roger
Published: June 2004

The protocols in this unit describe methods for preparing bacterial plasmid DNA free from chromosomal DNA. The first is an alkaline lysis miniprep suitable for screening a moderate number of bacterial colonies by restriction endonuclease cleavage and agarose gel electrophoresis. The second is the first step to producing large amounts (milligrams) of plasmid DNA and is also based on alkaline lysis of the bacterial cells. The crude lysate generated in this protocol can be further purified by centrifugation using CsCl/ethidium bromide (CsCl/EtBr) equilibrium density gradients. Three support protocols provide information on how to grow overnight and larger cultures of bacteria, and how to monitor bacterial growth with a spectrophotometer. Other methods, some relying on commercially available ion‐exchange columns, are discussed in the commentary.
Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
Authors: Gallagher Sean R.
Published: June 2004

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in molecular biology. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques are presented below. These three procedures cover a range from 5 to 10 ng/ml DNA to 50 mg/ml DNA.
Agarose Gel Electrophoresis
Authors: Voytas Daniel
Published: June 2004

This appendix presents a protocol for separating and purifying DNA fragments between 0.5 and 25 kb. A support protocol describes the use of midigels and minigels.
Palladium‐Mediated C5 Substitution of Pyrimidine Nucleosides
Authors: Ahmadian Mohammad , Klewer Douglas A. , Bergstrom Donald E.
Published: June 2004

One of the most efficient ways to link a reporter group to oligonucleotides is through the incorporation of a modified nucleoside during automated oligonucleotide synthesis. To be useful, it is important that the reporter group not interfere in hybridization reactions. This unit describes two linkers that can be used for the incorporation of a reporter group at the C5 position of deoxyuridine: a flexible aminoethylthioether linker, and a rigid amidopropynyl linker. The latter is suffciently long and positioned so that the reporter group lies outside the major groove of the DNA duplex.
Enzymatic Synthesis of M1G‐Deoxyribose
Authors: Schnetz‐Boutaud Nathalie C. , Chapeau Marie‐Christine , Marnett Lawrence J.
Published: June 2004

Adducts formed between electrophiles and nucleic acid bases are believed to play a key role in chemically induced mutations and cancer. M1G‐dR is an endogenous exocyclic DNA adduct formed by the reaction of the dicarbonyl compound malondialdehyde with a dG residue in DNA. It is an intermediate in the synthesis of a class of modified oligodeoxyribonucleotides that are used to study the mutagenicity and repair of M1G. This unit presents methods for synthesizing M1G‐dR by enzymatic coupling.
Unnatural Nucleosides with Unusual Base Pairing Properties
Authors: Bergstrom Donald E.
Published: June 2004

Synthetic modified nucleosides designed to pair in unusual ways with natural nucleobases have many potential applications in nucleic acid biochemistry. This overview lays the foundation for future protocol units on synthesis and application of unnatural bases, with particular emphasis on unnatural base analogs that mimic natural bases in size, shape, and biochemical processing. Topics covered including base pairs with alternative H‐bonding schemes, hydrophobic base pairs, degenerate bases, universal nucleosides, and triplex constituents.
Deoxyribonucleoside Phosphoramidites
Authors: Wilk Andrzej , Grajkowski Andrzej , Chmielewski Marcin K. , Beaucage Serge L. , Phillips Lawrence R.
Published: June 2004

The detailed preparation of deoxyribonucleoside phosphoramidites bearing a 4‐[N‐methyl‐N‐(2,2,2‐trifluoroacetyl)amino]butyl group for P(III) protection is presented. The use of this group circumvents nucleobase alkylation during oligonucleotide deprotection. Two syntheses of phosphoramidites starting from either a phosphordichloridite precursor or a bis‐(N,N‐diisopropylamino)chlorophosphine intermediate are described for the phosphinylation of suitably protected deoxyribonucleosides.
Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribo‐ and Oligoribonucleotides According to the H‐Phosphonate Method
Authors: Strömberg Roger , Stawinski Jecek
Published: June 2004

Oligonucleotides can be synthesized by condensing a protected nucleoside H‐phosphonate monoester with a second nucleoside in the presence of a coupling agent to produce a dinucleoside H‐phosphonate diester. This can then be converted to a dinucleoside phosphate or to a backbone‐modified analog such as a phosphorothioate or phosphoramidite. This unit discusses four alternative methods for synthesizing nucleoside H‐phosphonate monoesters. The methods are efficient and experimentally simple, and use readily available reagents. The unit describes the activation of the monoesters, as well as competing acylation and other potential side reactions.
Oligoribonucleotides with 2′‐O‐(tert‐Butyldimethylsilyl) Groups
Authors: Bellon Laurent
Published: June 2004

The chemical synthesis of oligoribonucleotides on solid support is routinely performed via the phosphoramidite method. However, the additional 2‐OH function of the ribofuranosyl sugar requires suitable protection during oligoribonucleotide synthesis. This unit describes methods for 2‐OH protection using the TBDMS group.
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

RNA 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

This 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

Locked 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

Locked 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

Protocols 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.
Biophysical Analysis of Nucleic Acids
Authors: Tinoco Ignacio
Published: June 2004

This overview unit provides a thorough overview of biophysical methods used for structure analysis, including X‐ray diffraction, nuclear magnetic resonance, optical spectroscopy, theoretical and computational methods, and single‐molecule methods. Advantages and disadvantages of the methods are compared.
NMR Determination of Oligonucleotide Structure
Authors: James Thomas L.
Published: June 2004

This 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 Folding Pathways
Authors: Crothers Donald M.
Published: June 2004

A general overview of the questions and problems in RNA folding is presented. Topics include the differences in the folding problems/questions that apply to RNA versus proteins, methods for determination of final structures, folding versus unfolding, resolution of space and time, and conformational switching.
RNA Secondary Structure Prediction
Authors: Mathews David H. , Turner Douglas H. , Zuker Michael
Published: June 2004

Details 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

Once 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

As 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

DNA 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

The 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

Modern 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

Imaging 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

Magnetic 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

This 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

Magnetic 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

Magnetic 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

This 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.
Spin and Gradient Echoes
Authors: Lin Weili
Published: June 2004

This unit discusses the basic concept of an echo. The phase of the spins plays the fundamental role in this process. A general description as to how an echo occurs is followed by a discussion on both spin echoes and gradient echoes.
Screening Forms for Patients for MR Procedures and Individuals for the MR Environment
Authors: Shellock Frank G.
Published: June 2004

This 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.
MR Safety Guide
Authors: Foster Glenn Jay , Augustine Lisa
Published: June 2004

This unit provides safety information for performing imaging sequences. Included are a checklist of common rules to abide by when performing MR imaging and an outline of the proper orientations required to be taken by staff members. In addition a guide thoroughly discussing important safety information is also provided.
Digestion of DNA with Restriction Endonucleases
Authors: Bloch Kenneth D. , Grossmann Barbara
Published: June 2004

Restriction 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

In 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

This 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

T4 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

With 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

Genomic 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

Enzymatic 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

The 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

In 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

This 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

This 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

Standard 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.
Denaturing Gel Electrophoresis for Sequencing
Authors: Slatko Barton E. , Albright Lisa M.
Published: June 2004

The accuracy of DNA sequence determination depends largely upon resolution of the sequencing products in denaturing polyacrylamide gels. This unit provides a detailed description of the setup, electrophoresis, and processing of such gels. In general, the gels required for DNA sequencing are 40‐cm long, of uniform thickness, and contain 4% to 8% acrylamide and 7 M urea. Modifications of this protocol increase the length of readable sequence information which can be obtained from a single gel (i.e., forming the gel with wedge‐shaped spacers to create a field gradient, or incorporating a buffer gradient, an electrolyte gradient, or an acrylamide step gradient into the gel). A modification to the ‐‐inclusion of formamide in the sequencing gel‐‐is designed to overcome gel compressions arising from secondary structure in the sequencing products during gel electrophoresis. A discussion of acrylamide concentrations and electrophoresis conditions is included in the Commentary.
Linker‐Scanning Mutagenesis of DNA
Authors: Greene John M.
Published: June 2004

Two protocols are described in which clusters of point mutations are introduced throughout a sequence of interest that has been cloned into a plasmid vector. The first protocol uses complementary oligonucleotides and requires a unique restriction site adjacent to the region that is to be mutagenized. A nested series of deletion mutations is first generated in the region. A pair of complementary oligonucleotides are synthesized to fill in the gap in the sequence of interest between the linker at the deletion endpoint and the nearby restriction site. The linker sequence actually provides the desired clusters of point mutations as it is moved or “scanned” across the region by its position at the varied endpoints of the deletion mutation series. An makes use of site‐directed mutagenesis procedures to introduce smaller clusters of point mutations throughout the target region.
Calcium Phosphate Transfection
Authors: Kingston Robert E. , Chen Claudia A. , Rose John K.
Published: June 2004

This unit presents two methods of calcium phosphate‐based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES‐buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency. In the alternate high‐efficiency method, a BES‐buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, both protocols yield similar results for transformation with linear plasmid or genomic DNA, or for transient expression.
Nonisotopic Assays for Reporter Gene Activity
Authors: Brasier Allan R. , Fortin John J.
Published: June 2004

This unit describes two nonisotopic systems for reporter gene activity in cells transfected with the firefly luciferase expression plasmid or the β‐galactosidase expression plasmid. Both of these chemiluminescent assays have the advantages of high sensitivity and broad linear range. In the chemiluminescent detection procedures given in this unit, both luciferase activity and β‐galactosidase activity can be measured with either a luminometer or a scintillation counter.
One‐Dimensional Electrophoresis Using Nondenaturing Conditions
Authors: Gallagher Sean R.
Published: June 2004

Nondenaturing or “native” electrophoresis (i.e., electrophoresis in the absence of denaturants such as detergents and urea) is an often‐overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Because mobility depends on the size, shape, and intrinsic charge of the protein, nondenaturing electrophoresis provides a set of separation parameters distinctly different from mainly size‐dependent denaturing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and charge‐dependent isoelectric focusing. Two protocols are presented in this unit. Continuous PAGE is highly flexible, permitting cationic and anionic electrophoresis over a full range of pH. The discontinuous procedure is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration.
Immunoblotting and Immunodetection
Authors: Gallagher Sean , Winston Scott E. , Fuller Steven A. , Hurrell John G.R.
Published: June 2004

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS‐PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody‐antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti‐IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.
Protein Identification and Characterization by Mass Spectrometry
Authors: Patterson Scott D.
Published: June 2004

This overview describes some of the new technologies that can be employed to facilitate rapid identification and characterization of proteins, including the use of correlative approaches for protein identification, rapid posttranslational modification analysis, identification of components in complex mixtures, and direct mass analysis of gel‐separated proteins. The mass spectrometric methods referred to in this overview include matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) and electrospray ionization mass spectrometry (ESI‐MS).
Purification of Monoclonal Antibodies
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R.
Published: June 2004

The uses of monoclonal antibodies as enzyme conjugates and as immunoaffinity reagents require their purification from the crude ascites fluid. This unit provides protocols for purification using protein A‐Sepharose chromatography and affinity chromatography, which are superior to ammonium sulfate precipitation, gel filtration, or ion‐exchange chromatography for the preparation of contaminant‐free antibody.
Purification of Immunoglobulin G Fraction from Antiserum, Ascites Fluid, or Hybridoma Supernatant
Authors: Cooper Helen M. , Paterson Yvonne
Published: June 2004

This unit describes the isolation of the immunoglobulin G (IgG) fraction (containing antibodies of all specificities) from a complex protein mixture such as antiserum, ascites fluid, or hybridoma supernatant. The basic procedure utilizes saturated ammonium sulfate solution to precipitate the IgG fraction, while an alternate protocol describes fractionation of IgG by chromatography on DEAE‐Affi‐Gel Blue resin.
Introduction to Peptide Synthesis
Authors: Fields Gregg B.
Published: June 2004

This overview presents a review of classical and current solid‐phase peptide synthesis techniques. Peptide synthesis has become a more practical part of present‐day scientific research following the advent of solid‐phase techniques, and has practical commercial and scientific implications. This unit provides information on solid‐phase peptide‐synthesis methodology, offering descriptions of different supports, coupling reagents, and practical approaches to the synthesis of modified residues and structures. It also presents information concerning protein synthesis and the purification and analysis of synthetic peptides. This methodology has great implications for the design of synthetic peptides to serve in antibody production.
Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins
Authors: Grant Gregory A.
Published: June 2004

Antibodies that recognize intact proteins can be produced through the use of synthetic peptides based on short stretches of the protein sequence, without first having to isolate the protein. The procedure for selecting the antigenic sequence is relatively straightforward. This unit identifies ways to determine the best sequence and to chemically couple the synthetic peptide to a carrier protein to boost the immune response. Another method entails direct synthesis of a MAP covalent multimer of the simple peptide sequence. Peptides can also be coupled to carrier proteins through either their amino or carboxyl groups. Alternate protocols in this unit cover these scenarios. In addition, methods for assaying free sulfhydryl content and for reducing disulfide bonds in synthetic peptides are provided. Finally, once the coupling procedure has been performed, it is possible to determine the approximate degree of coupling by amino acid analysis, and a procedure for this is also provided.
Mobility Shift DNA‐Binding Assay Using Gel Electrophoresis
Authors: Buratowski Stephen , Chodosh Lewis A.
Published: June 2004

The DNA‐binding assay using nondenaturing polyacrylamide gel electrophoresis (PAGE) provides a simple, rapid, and extremely sensitive method for detecting sequence‐specific DNA‐binding proteins. Proteins that bind specifically to an end‐labeled DNA fragment retard the mobility of the fragment during electrophoresis, resulting in discrete bands corresponding to the individual protein‐DNA complexes. The assay described in this unit can be used to test binding of purified proteins or of uncharacterized factors found in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA‐binding proteins. Three additional protocols describe a competition assay using unlabeled competitor DNA, an antibody supershift assay, and multicomponent gel shift assays.