bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of Endcap Dimethoxytrityl Phosphoramidites for Endcapped Oligonucleotides
Authors: Pingle Maneesh R. , Ng Pei‐Sze , Xu Xiaolin , Bergstrom Donald E.
Published: June 2004

Endcaps may be either aromatic or aliphatic molecules that specifically cross‐link the 5′ end of one strand with the 3′ end of the complementary strand in a DNA duplex. Endcaps may be viewed as a replacement of the loop region nucleotides of a DNA hairpin, with the added advantage of increased thermal stability. An endcap is incorporated into the sequence during oligonucleotide synthesis. Three endcaps are described in this unit. The naphthalene diimide endcap prefers to base stack with GC base pairs. The terthiophene endcap has higher lipophilicity than the naphthalene diimide endcap and provides higher stability when stacked over an AT base pair. The 2,2′‐oxydiacetamide endcap provides lower enhancement in stability but a more rigid and well‐defined structure than the oligo(ethylene glycol) endcaps. Synthesis of endcapped oligonucleotides can be carried out using standard automated synthesis protocols with only minor modifications.
Engineering Terminal Disulfide Bonds Into DNA
Authors: Glick Gary D.
Published: June 2004

This protocol presents a simple and general means of modifying nucleic acids with disulfide cross‐links. These cross‐links serve as powerful tools to probe the structure, dynamics, thermodynamics, folding, and function of DNA and RNA, much in the way that cystine cross‐links have been used to study proteins. The chemistry described has been used to synthesize disulfide‐cross‐linked hairpins and duplexes, higher‐order structures such as triplexes, non‐ground‐state conformations, and tRNAs. since the cross‐links form quantitatively by mild air oxidation and do not purturb either secondary or tertiary structure, this modification should prove quite useful for the study of nucleic acids.
Probing RNA Structure with Chemical Reagents and Enzymes
Authors: Ziehler William A. , Engelke David R.
Published: June 2004

This unit provides thorough coverage of the most useful chemical and enzyme probes that can be used to examine RNA secondary and tertiary structure. Footprinting methods are presented using dimethyl sulfate, diethyl pyrocarbonate, ethylnitrosourea, kethoxal, CMCT, and nucleases. For chemical probes, both strand scission and primer extension detection protocols are included.
Probing DNA Structure with Hydroxyl Radicals
Authors: Tullius Thomas D.
Published: June 2004

The hydroxyl radical is a useful probe for studying the shape of the surface of a DNA molecule. Using this technique, fine details of DNA structure can potentially be revealed. This unit describes how to use the hydroxyl radical to generate a random cleavage pattern at the surface of the molecule, separate the broken DNA strands by polyacrylamide gel electrophoresis, and analyze the cleavage pattern to give an image of the surface of the molecule.
Optical Methods
Authors: Plum G. Eric
Published: June 2004

Equilibrium optical melting curves are used in this unit to determine thermodynamic parameters of nucleic acid complex formation. This section contains all of the relevant equations and a discussion of which are most appropriate to a given situation. Additionally, procedures are given for making preliminary determinations of molar extinction coefficients and for determining the number of oligonucleotides in a complex. The section on extinction coefficients is particularly essential to anyone needing to know solution concentrations.
Molecular Modeling of Nucleic Acid Structure: Electrostatics and Solvation
Authors: Cheatham Thomas E. , Brooks Bernard R. , Kollman Peter A.
Published: June 2004

This unit presents an overview of computer simulation techniques as applied to nucleic acid systems, ranging from simple in vacuo molecular modeling techniques to more complete all‐atom molecular dynamics treatments that include an explicit representation of the environment. The third in a series of four units, this unit focuses on critical issues in solvation and the treatment of electrostatics.
Molecular Modeling of Nucleic Acid Structure: Setup and Analysis
Authors: Cheatham Thomas E. , Brooks Bernard R. , Kollman Peter A.
Published: June 2004

The last in a set of units by these authors, this unit addresses some important remaining questions about molecular modeling of nucleic acids. It describes how to choose an appropriate molecular mechanics force field; how to set up and equilibrate the system for accurate simulation of a nucleic acid in an explicit solvent by molecular dynamics or Monte Carlo simulation; and how to analyze molecular dynamics trajectories.
Determination of Binding Mode: Intercalation
Authors: Dedon Peter C.
Published: June 2004

A small molecule can be assumed to bind to DNA by intercalation between base pairs if it causes lengthening and unwinding of the DNA helix and undergoes changes in its spectral properties, such as DNA‐induced hypochromism and quenching of its UV absorbance. DNA lengthening and unwinding can be determined from the change in viscosity of a solution of linear or plasmid DNA, respectively. Intercalation of a ligand can also be seen as a reduction in the UV/visible absorbance of the intercalator, as well as a shift in the absorbance maximum.
Chemistry of Minor Groove Binder–Oligonucleotide Conjugates
Authors: Kutyavin Igor , Lokhov Sergey , Lukhtanov Eugene , Reed Michael W.
Published: June 2004

Various types of minor groove binders have been attached to synthetic oligodeoxynucleotides, and the interactions of these conjugates (MB‐ODNs) with DNA are reviewed here. MB‐ODNs have enhanced DNA affinity and have improved the hybridization properties of sequence‐specific DNA probes. Short MB‐ODNs hybridize with ssDNA to give more stable DNA duplexes than unmodified ODNs with similar lengths. Mismatch discrimination of short MB‐ODNs is enhanced in comparison to longer unmodified ODNs. The stronger binding of MB‐ODNs allows for more stringent hybridization conditions to be used in DNA probe‐based assays. MB‐ODNs are especially useful in quantitative “real‐time” PCR assays since they bind efficiently during the high‐temperature primer extension cycle. The synthesis and biophysical chemistry of MB‐ODN conjugates are reviewed here. Four published structural classes of MB‐ODNs and their various dsDNA binding modes are discussed, and the well‐characterized DPI3‐type MB‐ODNs and their interactions with ssDNA target strands are described in detail.
Theoretical Principles of In Vitro Selection Using Combinatorial Nucleic Acid Libraries
Authors: Vant‐Hull Barry , Gold Larry , Zichi Dominic A.
Published: June 2004

A new paradigm for drug discovery and biological research has developed from technologies that integrate combinatorial chemistry with rounds of selection and amplification, a technique called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). This overview unit discusses nucleic acid libraries that can be used, affinity probability distributions, an equilibrium model for SELEX, and optimal conditions including concentrations and signal‐to‐noise ratios.
Design, Synthesis, and Amplification of DNA Pools for In Vitro Selection
Authors: Pollard Jack , Bell Sabine D. , Ellington Andrew D.
Published: June 2004

Preparation of a random‐sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer. Purification of a single‐stranded pool and conversion to a double‐stranded pool are presented as step‐by‐step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions.
In Vitro Selection of RNA Aptamers to a Small Molecule Target
Authors: Jhaveri Sulay , Ellington Andrew
Published: June 2004

This unit describes the selection of aptamers that bind to a small molecule target from a single‐stranded RNA pool. Aptamers generated from this type of selection experiment can potentially function as receptors for small molecules for applications ranging from medical diagnostics to environmental monitoring. This unit describes two modes of selection, one by column filtration and one by batch selection.
Analysis of Oxidized DNA Fragments by Gel Electrophoresis
Authors: Awada Mohamad , Dedon Peter C.
Published: June 2004

Polyacrylamide gel electrophoresis is used to define and quantify products of deoxyribose oxidation in DNA, based on the unique electrophoretic mobility of DNA fragments possessing deoxyribose oxidation products on their termini. This approach allows initial estimation of the chemistry. Once the chemical identity of damage products has been confirmed, this technique allows sensitive quantitation of the various damage products.
Probing RNA Folding Pathways by RNA Fingerprinting
Authors: Woodson Sarah A.
Published: June 2004

This unit provides protocols for using native polyacrylamide gel electrophoresis to distinguish folding and unfolding conformers of RNA. It is useful for studying conformers that can exchange in a period of minutes or seconds, and that are thus difficult to study by solution‐based methods. Conformers that have been separated and immobilized in the gel matrix can be used to studying catalytic activity with or without being eluted from the gel. The method can be applied to a wide varied of catalytic RNAs and RNA‐protein complexes.
Characterization of Tertiary Folding of RNA by Circular Dichroism and Urea
Authors: Sosnick Tobin R.
Published: June 2004

CD spectroscopy can be used to monitor RNA tertiary folding transitions that may not be observable by absorbance spectroscopy. With the use of computer‐controlled titrators, data can be acquired rapidly, and accurate thermodynamic properties can be obtained over a wide variety of conditions. Thus, CD spectroscopy provides a useful complement to site‐resolved or chemical modification methods.
Time‐Resolved Hydroxyl Radical Footprinting of RNA with X‐Rays
Authors: Woodson Sarah A. , Deras Michael L. , Brenowitz Michael
Published: June 2004

RNA tertiary structure and protein interactions can be detected by protection from hydroxyl radical cleavage. Generation of hydroxyl radicals with a synchrotron X‐ray beam provides analysis on a short timescale (50 msec to 100 sec), which enables the structures of folding intermediates or other transient conformational states to be determined. This unit provides detailed instructions on the use of the synchrotron beamline for hydroxyl radical footprinting.
Use of Fluorescence Spectroscopy to Elucidate RNA Folding Pathways
Authors: Bevilacqua Philip C. , Turner Douglas H.
Published: June 2004

This overview unit discusses fluorescence spectroscopy as a tool for studying RNA folding. Ribozymes and oligonucleotides can be labeled with a fluorescent probe and analyzed to give information about both slow and fast kinetic processes with real‐time data acquisition. The unit discusses the advantages and disadvantages of various pendant probes and nucleotide analogs, the analytical methods that can be used, instrument setup, control experiments, and a variety of kinetic experiments that can be performed, such as determination of rate constants.
Probing RNA Structural Dynamics and Function by Fluorescence Resonance Energy Transfer (FRET)
Authors: Walter Nils G.
Published: June 2004

Biological function of RNA is often mediated by cyclic switching between several (meta‐)stable arrangements of tertiary structure. Fluorophore labeling of RNA offers a unique view into these folding and conformational switching events, since a fluorescence signal is sensitive to its molecular environment and can be continuously monitored in real time to produce kinetic rate information. This unit focuses on the practical implications of using fluorescence resonance energy transfer (FRET) to probe RNA structural dynamics and function. FRET is a particularly powerful fluorescence technique since, in addition to kinetic data, it provides insights into the structural basis of a conformational rearrangement. Protocols describe how to postsynthetically label RNA for FRET and how to acquire and analyze FRET data. Support protocols describe methods for deprotecting synthetic RNA and for purifying RNA by gel electrophoresis and HPLC. Considerations for selecting appropriate RNA, fluorophores, and labeling strategies are discussed in detail in the commentary.
Overview of the Synthesis of Nucleoside Phosphates and Polyphosphates
Authors: Johnson David C. , Widlanski Theodore S.
Published: June 2004

This overview summarizes methodology used for the synthesis of nucleoside mono‐, di‐, and triphosphates. Selected techniques such as the Mitsunobu reaction, displacement reactions involving nucleoside 5′‐tosylates, “anion‐exchange” techniques, and phosphoramidite and phosphoramidate methodologies are highlighted. The chemistry of phosphorylation is detailed with respect to advantages and limitations under various conditions. Applicability of the methods toward the synthesis of analogs such as imidophosphates, phosphorothioates, and radiolabeled nucleotides is also addressed.
Characteristics of Nucleic Acids
Authors: Bergstrom Donald E.
Published: June 2004

This appendix is a compilation of information from Current Protocols in Molecular Biology (John Wiley & Sons, Inc.) and provides information about the major nucleoside/nucleotide constituents and structures for A‐, B‐, and Z‐DNA. Tables and figures provide (1) experimentally useful properties of the nucleoside/nucleotide building blocks; (2) chemical structures of the nucleosides; (3) aspects of nucleotide stereochemistry that are important to an understanding of base pairing and secondary structure; (4) alternative (non‐Watson‐Crick) base pairings; and (5) the three best characterized helix structures for DNA: A‐, B‐, and Z‐DNA.
Thin‐Layer Chromatography
Authors: Meyers C.L.F.
Published: June 2004

TLC is used extensively in nucleic acid chemistry to monitor the progress of chemical reactions, to assay fractions collected from a larger chromatographic separation (e.g., column chromatography), and to determine optimal conditions prior to column chromatography. This unit describes methods for spotting test compounds onto a TLC plate, developing the plate in a suitable solvent system, visualizing the results, and calculating the retention factor (Rf). Candidate compounds can be co‐spotted for identification without relying on Rf values.
Extracranial Carotid Artery Disease
Authors: Haacke E. Mark , Kido Daniel , Tong Karen
Published: June 2004

Magnetic Resonance Angiography (MRA) is a noninvasive means for studying the integrity of the vascular system. This unit presents a basic protocol describing an MRA technique for imaging the carotid arteries, time‐of‐flight MRA, and an for contrast‐enhanced MRA. A second basic protocol based on high‐resolution spin‐echo scanning is also described. This protocol can be used to study the vessel wall in more detail. The parameters given here are derived from experience at 1.5 T and may need to be altered slightly depending on the field strength and the equipment manufacturer. In particular, optimal TE may vary with different field strength.
Imaging Venous Angiomas
Authors: Midyett F. Allan , Mukherji Suresh , Fisher Laurie
Published: June 2004

Chapter A2.3 discusses non‐invasive cerebral venous imaging for those patients thought to have venous angiomas using Magnetic Resonance Venography (MRV) and MRI. Use of specific protocols is described.
Metastatic Intra‐Axial Neoplasia
Authors: Nusbaum Annette O. , Atlas Scott W.
Published: June 2004

The role of MRI imaging in the metastatic work‐up is to detect the spread of tumor to the brain parenchyma, and define the location. Intravenous contrast (gadolinium‐DTPA) provides the greatest sensitivity for detecting brain lesions and is almost always indicated except when there is no intravenous access. This unit presents a for imaging intra‐axial brain metastases and specific modifications are discussed for the case of metastatic brain tumors demonstrating evidence of hemorrhage. The sequences described in this unit are based on 1.5 T scanner (Echospeed GE Medical Systems, Milwaukee, Wisconsin), but can be expected to be equally applicable to other field strengths and scanners from other manufacturers.
Metastatic Extra‐Axial Neoplasia
Authors: Nusbaum Annette O. , Atlas Scott W.
Published: June 2004

There are a multitude of tumors that may spread to the extra‐axial compartment (i.e., meninges, skull base, and calvarium). The role of imaging in the search for metastases is to detect the full extent of and to correctly localize the disease. This unit presents basic protocols for imaging extra‐axial metastatic tumors. Specific modifications are discussed where necessary. The sequences described in this unit are based on a 1.5 T scanner (Echospeed GE Medical Systems, Milwaukee, Wisconsin), but can be expected to be equally applicable to other field strengths and scanners from other manufacturers.
Extra‐Axial Tumors
Authors: Nusbaum Annette O. , Atlas Scott W.
Published: June 2004

Extra‐axial tumors most commonly arise from the meninges, calvarium, or skull base. Localization of a lesion as extra‐axial or extra‐cerebral in origin has significant clinical importance in terms of treatment planning and predicting prognosis. High‐resolution fat‐suppressed imaging is crucial for specific evaluation of the skull base, and correlation with computed tomography (CT) is often helpful in these cases. This unit presents basic MRI protocols for imaging extra‐axial tumors in specific locations; sequence modifications are discussed where necessary. The sequences described in this unit are based on a 1.5 T scanner (GE Medical Systems), but can be expected to be equally applicable to other field strengths and scanners from other manufacturers.
Meningitis
Authors: Auber Andrew E. , Belden Clifford
Published: June 2004

Magnetic Resonance Imaging (MRI) in meningitis, as with most other forms of intracranial inflammatory or infectious diseases, is a powerful though largely non‐specific diagnostic tool. The technique is used to detect the presence of disease, and also complications related to the primary process, as well as assess for alternative diagnoses. For imaging these patients, a standard imaging protocol is utilized which includes gadolinium‐enhanced sequences. This unit presents a protocol based on diffusion MRI (dMRI), which can be employed if specific clinical situations require further clarification. The parameters given in this unit are derived from experience at 1.5T and may need to be altered slightly depending on the field strength available and the specific equipment manufacturer.
Clinical Applications of fMRI
Authors: Thulborn Keith R. , Davis Denise
Published: June 2004

The clinical scenario in which fMRI is requested by a referring physician is usually after detection of a lesion in the brain of a patient for whom surgical intervention is being contemplated. This unit discusses the suitable parameters of a functional MRI system and also presents the for imaging language function.
Cranial Nerves IX To XII
Authors: Evers Robert W. , Yousem David M.
Published: June 2004

Cranial nerves IX to XII are rarely affected by pathology compared with cranial nerves III, V, VII, and VIII. Nonetheless, their evaluation is challenging, since lesions of these nerves span the gamut from intracranial to extracranial sites. Imaging of these cranial nerves requires a focused approach based on clinical symptomatology and signs. This unit presents the basic protocol for imaging cranial nerves IX to XII. An alternate protocol is presented for cases where non‐neoplastic lesions are considered.
The Orbit and Optic Nerves
Authors: Chakeres Donald William , Bourekas Eric C.
Published: June 2004

Magnetic Resonance Imaging (MRI) of the orbits and optic nerves has developed into the “gold standard” of imaging modalities for the evaluation of many soft tissue abnormalities of the orbit. Computed tomography (CT) remains the modality of choice for evaluation of the bony structures of the orbit. MRI is also more flexible, allowing for multiplanar imaging that is not possible with CT. This unit presents a basic protocol for imaging of the orbit. An alternate protocol is presented for the case of dedicated high‐resolution surface coil orbital study.
Herniated Intervertebral Disc
Authors: Jinkins J. Randy , Stark David D.
Published: June 2004

One of the most significant impacts of magnetic resonance imaging (MRI) has been its ability to exquisitely depict normal and pathologic anatomy of the spine. This unit presents a basic protocol for conventional fast spin echo imaging of the spine. An alternate protocol is presented for gradient recalled echo acquisitions that may be used in the sagittal and/or transverse planes to clearly distinguish between discs and soft tissue and to clarify the spinal neural foramen in the cervical region. A second alternate protocol is presented for contrast enhanced MRI acquisitions.
Spinal Canal Stenosis
Authors: Jinkins J. Randy , Stark David D.
Published: June 2004

This unit presents a basic protocol for fast spin echo sequences for imaging to diagnose stenosis of the spinal canal. Spinal stenosis is defined generally as a narrowing of one or more of the following: the central canal, foramina, and lateral recesses of the lumbar spine. Specifically, this stenosis can be classified into three types as defined by location: (a) stenosis of the central spinal canal, (b) stenosis of the intervertebral spinal neural foramen or foramina, and (c) stenosis of the lateral recess(es) of the central spinal canal.
Spondylosis Deformans
Authors: Jinkins J. Randy , Stark David D.
Published: June 2004

This unit presents a basic protocol of conventional and fast spin echo acquisition for detecting spondolysis deformans. The margins of the osteophytosis associated with spondylosis deformans are generally well defined utilizing fast spin echo acquisitions. An alternate protocol is presented for gradient recalled echo acquisitions that may be used in the sagittal and/or transverse planes to clearly distinguish between discs and soft tissue, and to clarify the spinal neural foramen in the cervical region.
Spinal Inflammation
Authors: Jinkins J. Randy , Stark David D.
Published: June 2004

This unit presents a basic protocol for conventional and fast spin echo imaging of spine for evaluation of infections involving the CNS. The epidemic of acquired immunodeficiency syndrome (AIDS) and the frequency of CNS infections has been on the rise. An alternate protocol is presented for coronal acquisition that may be helpful to analyze the perispinal tissues for abscess formation.
Extradural Spinal Cord/Cauda Equina Compression
Authors: Jinkins J. Randy , Stark David D.
Published: June 2004

Clinical signs and symptoms suggesting acute or subacute compression of the spinal cord or cauda equina constitute a medical emergency requiring urgent diagnosis in order to effect appropriate therapy for alleviating the pathologic process responsible for the compressive phenomenon. The purpose of MR imaging in such cases is to determine the level(s), degree, and, if possible, the type of disease process in order to assist in therapeutic planning gauged toward relieving the neurologic compression. This unit presents a basic protocol for conventional and fast spin echo imaging of spine for such cases. Two alternate protocols are presented for cases where it is necessary to distinguish between the spinal cord and the extradural tissue comprising this structure.
Post Surgical Spinal Evaluation
Authors: Jinkins J. Randy , Stark David D.
Published: June 2004

One of the most challenging areas of diagnosis is to be found in acquiring and interpreting medical images in the patient who has undergone lumbosacral surgery for spinal degenerative disease. The section illustrates the features of expected and abnormal postsurgical spinal imaging. These discussions provide the background, practical information, and graphic examples necessary to enable the medical imaging physician to better approach the clinicoradiologic evaluation of the postsurgical patient.
Intramedullary Spine Disease
Authors: Bradley William G.
Published: June 2004

One of the most significant impacts of magnetic resonance (MRI) has been its ability to exquisitely depict normal and pathologic anatomy of the spine. This unit presents a that deals with intramedullary disease, i.e., that involving the cord. An is presented for cases of extramedullary‐intradural disease.
Pericardial Disease
Authors: Lee Vivian S.
Published: June 2004

This unit presents a basic technique for evaluating non‐neoplastic disease of the pericardium with optional contrast‐enhanced sequences for evaluating suspected neoplastic disease. The parameters are based on experience on a Siemens 1.5 T Vision or Symphony and should be altered accordingly for different field strengths and machines from different manufacturers.
Cardiac Masses
Authors: Lee Vivian S.
Published: June 2004

Magnetic Resonance Imaging (MRI) may be indicated in patients who have poor acoustic windows or in whom lesions are incompletely characterized by echocardiography. This unit presents a basic technique for evaluating cardiac masses, with optional contrast‐enhanced sequences for specific indications. The parameters are based on experience on a Siemens 1.5 T Vision or Symphony and should be altered accordingly for different field strengths and machines from different manufacturers.
Aortic Aneurysm and Pseudoaneurysm Assessment
Authors: Contreras Maricela , Amin Rajesh S. , Yucel E. Kent
Published: June 2004

Although contrast angiography is still considered the “gold standard” for evaluation of the aorta and its major branches, Magnetic Resonance Angiography (MRA) has quickly gained popularity as an imaging tool for the assessment of the entire aorta. MRA serves as an alternative imaging modality that can be utilized in patients with impaired renal function and with allergies to iodinated contrast medium (iodinated contrast medium is required in contrast angiography and computed tomography, CT). The purpose of this unit is to present fundamental MRA techniques useful in the evaluation of the thoracic and abdominal aorta based on experience on a 1.5 T GE LX scanner.
Pulmonary Embolism
Authors: Bis Kostaki G. , Shetty Anil N.
Published: June 2004

There have been a number of approaches taken to image the pulmonary vasculature. This unit presents basic protocols based on black blood spin echo and/or gradient echo techniques for detection of pulmonary embolisms and deep vein thrombosis. Bright blood magnetic resonance angiography (MRA), 2‐D time‐of‐flight (TOF), and 3‐D contrast‐enhanced MRA is also presented for visualizing the entire vascular tree. The parameters provided in this unit are acquired from Siemens 1.5T Vision Scanner. These parameters may need to be altered depending on the field strength and equipment manufacturer.
Deep Vein Thrombosis Detection
Authors: Bis Kostaki G. , Shetty Anil N.
Published: June 2004

Magnetic Resonance Angiography (MRA) is a robust and noninvasive technique for evaluating the vascular system with respect to deep venous thrombosis (DVT) of the lower extremities. This unit presents basic protocols for imaging the pelvic and lower extremity veins for evaluating DVT. A protocol is also provided for defining the sequence for imaging the pelvic and lower extremity veins with a stepping table.
Brachial Plexus
Authors: Takahashi Naoki , Narra Vamsidhar
Published: June 2004

MRI is the primary imaging modality used to diagnose the cause of brachial plexopathy. This unit presents protocols to diagnose the cause of brachial plexus. The provides the core component of this procedure and an alternate protocol is presented for the case of avulsion injury from trauma. A second alternate protocol is presented for the case where neoplastic involvement of the brachial plexus is suspected or any abnormalities are seen. A third alternate protocol is presented for the cases when the patient has symptoms of thoracic outlet syndrome and vascular compression is suspected.
MRI of the Female Pelvis
Authors: Wilber Kathy , Semelka Richard , Fisher Laurie
Published: June 2004

MRI provides an effective means for evaluating the female pelvis. This unit present a basic protocol for imaging the female pelvis. The protocol is designed to evaluate for the presence of fibroids or adenomyosis, uterine anomalies, and endometriosis. This protocol also provides limited evaluation of adnexal disease processes including: identification of ovaries, hemorrhagic ovarian cysts, and dermoid tumors.
Ovarian Cancer Diagnosis and Staging
Authors: Mitchell Donald G. , Natale Peter , Holland George
Published: June 2004

This unit presents a basic protocol for detecting and staging ovarian cancer. The techniques are similar to those used for general imaging of the liver/abdomen and the pelvis for other applications.
Silicone Breast Implants
Authors: Gorczyca David P.
Published: June 2004

Magnetic resonance imaging is a powerful noninvasive means by which to study the integrity of silicone breast implants. Silicone has a unique MR frequency and long T1 and T2 relaxation times that allow for several MR sequences to provide excellent diagnostic images. This unit presents a basic protocol for imaging of silicon breast implants.
Rotator Cuff Disease
Authors: Gigena Leopoldo M. , Chung Christine B. , Resnick Donald
Published: June 2004

Shoulder pain is a common clinical presentation. Though the spectrum of disorders affecting the shoulder is quite diverse, emphasis is often placed on abnormalities of the rotator cuff and glenohumeral joint instability. The rotator cuff plays an important role in both the function and stability of the shoulder. Moreover, its pathology, with early and accurate diagnosis, is amenable to treatment, which can markedly affect patient outcome. This unit presents an MR imaging protocol for evaluation of the glenohumeral joint. Though specific emphasis is placed on the rotator cuff, it can also be used for global assessment of the shoulder.
MR Arthrography of Shoulder
Authors: Chung Christine B. , Gigena Leopoldo M. , Resnick Donald
Published: June 2004

The glenohumeral joint boasts the greatest range of motion of any peripheral joint in the body, but not without cost; it is also the most frequently dislocated joint in the body. Stability of this articulation is limited for two major reasons. The articulating surface of the glenoid is significantly smaller than that of the humeral head, and the joint capsule is redundant and provides little support. This unit presents a basic MR arthrography protocol for evaluation of glenohumeral joint instability.
Bone Stress Injuries
Authors: Bergman Gabrielle , Jones Timothy R.
Published: June 2004

This unit outlines basic MRI protocols for evaluating the most common bone stress injuries including those of the tibia, femoral neck, femoral shaft, metatarsal, and navicular bone. The protocols concentrate on the lower extremity, as the majority of stress injures occur in this region. These protocols were developed using a 1.5 T system (Signa, General Electric Medical Systems). However, the sequences described could be adapted to low‐ or mid‐field scanners using STIR (short tau inversion recovery) sequences to acquire the T2‐weighted images. This technique would thus require longer scan times and lower resolution matrices.
Abnormalities of the Osseous Structures of the Hip and Peri‐Articular Soft Tissues
Authors: Kneeland J. Bruce , Kaut Roth Carolyn , Garrison James , Testa Anthony
Published: June 2004

This unit presents a basic protocol for detection and assessment of a large number of disorders arising in the bones of the hip or the surrounding soft tissues, including osteonecrosis, transient osteopenia, fractures, soft tissue injuries, and tumors. All of these disorders can be readily assessed without the use of a contrast agent. The evaluation of intra‐articular structures, in particular the acetabular labrum, however, is probably best accomplished with the use of the direct injection of a Gd‐chelate contrast agent into the hip joint. The protocol is presented for a 1.5‐Tesla system for evaluation of disease in the region of the hip. Modifications for low‐field (0.23 to 0.3 T) systems are also discussed.
Magnetic Resonance Imaging of the Gastrointestinal Tract
Authors: Low Russell N.
Published: June 2004

MRI provides comprehensive information on gastrointestinal diseases, including infectious, inflammatory, and malignant processes. We employ a set protocol incorporating breath‐hold SS RARE imaging combined with gadolinium‐enhanced, fat‐suppressed SPGR imaging in the transverse and coronal planes. Bowel distension is accomplished with inexpensive water‐soluble intraluminal contrast agents.
Short‐TR, Coherent, Gradient Echo Imaging
Authors: Norman Cheng Yu‐Chung , Haacke E. Mark
Published: June 2004

When a spin system is repeatedly disturbed by a fast repetition of RF pulses, the transverse magnetization after each new RF pulse approaches a steady‐state value which is smaller than the thermal equilibrium value. The spin system takes a finite number of pulses before this steady‐state is reached in a time that depends on both the T1 of the tissue and the flip angle of the RF pulse. The focus of this unit is on understanding the build‐up of the magnetization to steady‐state and the practical implementation of the simplest forms of imaging in the steady‐state. Sequences utilizing a steady‐state approach can be broadly classified as steady‐state coherent (SSC) and steady‐state incoherent (SSI) sequences. The SSC behavior is the subject of this unit.
Contrast
Authors: Celik Azim , Lin Weili
Published: June 2004

Contrast (Azim Celik, General Electric Company, Milwaukee, Wisconsin and Weili Lin, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina). SNR (signal‐to‐noise ratio) determines the effectiveness of an imaging experiment. However, even the highest SNR does not guarantee the usefulness of an image. An important aim of imaging for diagnostic purposes is to be able to distinguish between diseased and neighboring normal tissues. If the imaging method used does not have a signal‐manipulating mechanism which produces different signals for the diseased and normal tissues, then distinguishing the two tissues is not possible. This unit provides a detailed discussion of the contrast‐producing mechanisms that arise from the signal dependence on a wide variety of tissue parameters.
Contrast Agents
Authors: Celik Azim , Lin Weili
Published: June 2004

Contrast agents in general are exogeneous substances employed to alter natural tissue contrast. The motivation behind the use of contrast agents in MR imaging is to further enhance contrast between normal and diseased tissue types and indicate functionality of an organ. The focus of this unit is to give to the basic mechanisms of contrast agents in MR without going into their clinical applications.
Diffusion Tensor MR Imaging
Authors: Chenevert Thomas L. , Welsh Robert C.
Published: June 2004

This unit reviews the physical principles and methodologies involved in diffusion‐weighted imaging (DWI) and diffusion tensor imaging (DTI) for clinical applications. Diffusion‐sensitive MRI noninvasively provides insight into processes and microscopic cellular structures that alter molecular water mobility. Formalism to extend the Bloch equation to include effects of random translational motion through field gradients is reviewed. Definition of key acquisition parameters is also reviewed along with common methods to calculate and display tissue diffusion properties in a variety of image formats. Characterization of potential directional‐dependence of diffusion (i.e., anisotropy), such as that which exists in white matter, requires DTI. Diffusion tensor formalism and measurement techniques then reduce the diffusion tensor into standard anisotropy quantities that are summarized along with commonly used methods to depict directional information in an image format.
Large‐Scale Preparation of Plasmid DNA
Authors: Heilig J.S. , Elbing Karen L. , Brent Roger
Published: June 2004

Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high‐quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion‐exchange chromatography, and size‐exclusion chromatography.
Introduction to Lambda Phages
Authors: Lech Karen , Brent Roger
Published: June 2004

The biology of lambda‐derived vectors is extremely well understood. This introduction to the biology of lambda and related phages is included to help readers use the lambda vectors that are employed in the manual and to help readers understand new lambda vectors that are being developed. Specific topics include a discussion of lytic growth (i.e., requirements for DNA replication, packaging and lysis) and lysogenic growth (i.e., requirements for lysogenization and induction of a lysogen).
Lambda as a Cloning Vector
Authors: Lech Karen , Brent Roger , Irwin Nina
Published: June 2004

Advantages of lambda as a cloning vector are discussed along with considerations for the insert DNA (i.e., size, spi and hfl state). Maps of lambda‐derived cloning vectors are provided in addition to a discussion and map of a cosmid (a lambda‐derived plasmid vector).
Growing Lambda‐Derived Vectors
Authors: Lech Karen
Published: June 2004

It is often necessary to grow large quantities of lambda‐derived vectors, so that DNA can be made from them. This unit describes a procedure for making a phage stock by plate lysis, and an gives details on how to make a liquid lysate. Storage of phage lysates is described in a .
Introduction to Vectors Derived from Filamentous Phages
Authors: Greenstein David , Brent Roger
Published: June 2004

Many vectors in current use are derived from filamentous phages. These vectors are used because DNA inserted into them can be recovered in two forms: double‐stranded circles and single‐stranded circles. This overview unit describes the lifecycle of filamentous phages along with the development and use of filamentous phage vectors.
Purification and Concentration of DNA from Aqueous Solutions
Authors: Moore David , Dowhan Dennis
Published: June 2004

This unit presents basic procedures for manipulating solutions of single‐ or double‐stranded DNA through purification and concentration steps. These techniques are useful when proteins or solute molecules need to be removed from aqueous solutions, or when DNA solutions need to be concentrated. The , using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for purification of DNA from small volumes (<0.4 ml) at concentrations lower than 1 mg/ml. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads and this is also presented. These protocols may also be used for purifying RNA. The final two alternate protocols are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing low‐molecular‐weight oligonucleotides and triphosphates.
Purification of DNA by Anion‐Exchange Chromatography
Authors: Budelier Kim , Schorr Joachim
Published: June 2004

Column chromatography has evolved to provide a rapid and effective alternative to more laborious methods for preparing high‐quality DNA, such as CsCl‐gradient centrifugation. This unit describes the use of a column made of a unique anion‐exchange resin that selectively binds nucleic acids, allowing rapid separation of DNA from contaminating RNA, proteins, carbohydrates, and metabolites. The procedure employs columns supplied by QIAGEN; other preparation methods are available from other suppliers. A crude nucleic acid sample (usually a cleared cell lysate) is applied to the QIAGEN tip under conditions that favor binding. Contaminants in the sample are washed from the column with a moderate‐salt buffer, and DNA is eluted using a high‐salt buffer.
Agarose Gel Electrophoresis
Authors: Voytas Daniel
Published: June 2004

Agarose gel electrophoresis is a simple and highly effective method for separating, identifying, and purifying 0.5‐ to 25‐kb DNA fragments. The protocol can be divided into three stages: (1) a gel is prepared with an agarose concentration appropriate for the size of DNA fragments to be separated; (2) the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation; and (3) the gel is stained or, if ethidium bromide has been incorporated into the gel and electrophoresis buffer, visualized directly upon illumination with UV light.
Pulsed‐Field Gel Electrophoresis
Authors: Finney Michael
Published: June 2004

DNA molecules longer than 25 kb are poorly resolved by standard agarose gel electrophoresis. These longer molecules can be resolved using several techniques that periodically change the direction of the electric field in the gel. This unit describes the simplest and most generally useful of the pulsed‐field techniques, field inversion electrophoresis, which can be tuned to resolve molecules from ˜10 to 2000 kb (or more with specialized equipment). To resolve molecules beyond the range of field inversion, it is necessary to use some sort of field‐angle alternation electrophoresis such as CHEF (contour‐clamped homogeneous electric field; described in an ). A method is also provided for preparing high‐molecular‐weight DNA samples and size markers embedded in agarose blocks.
Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
Authors: Moore David , Dowhan Dennis , Chory Joanne , Ribaudo Randall K.
Published: June 2004

This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first protocol describes electroelution of the fragment of interest from standard agarose gels using buffer‐filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA‐45 paper (second protocol) provides relatively high yields for fragments <2000 bp. Fragments >1000 bp can also be separated on low gelling/melting agarose gels and purified by phenol extraction (third protocol), b‐agarase digestion of the gel (first alternate protocol), or via glass beads extraction (second alternate protocol). Removing linkers from a fragment using a column rather than a gel is described, followed by a method for estimating DNA concentrations in solution.
Southern Blotting
Authors: Brown Terry
Published: June 2004

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit presents several protocols for DNA transfer to nylon and nitrocellulose membranes, and for calibrating a UV transilluminator to cross‐link the transferred DNA to the membrane.
Dot and Slot Blotting of DNA
Authors: Brown Terry
Published: June 2004

Dot and slot blotting are simple techniques for immobilizing bulk unfractionated DNA on a nitrocellulose or nylon membrane. Hybridization analysis can then be carried out to determine the relative abundance of target sequences in the blotted DNA preparations. Dot and slot blots differ only in the geometry of the blot, a series of spots giving a hybridization pattern that is amenable to analysis by densitometric scanning. Samples are usually applied to the membrane using a manifold attached to a suction device. The describes such a procedure for dot or slot blotting on an uncharged nylon membrane; annotations to the steps detail the minor modifications that are needed if blotting onto nitrocellulose. The first alternate protocol describes the more major changes required for blotting with a positively charged nylon membrane. A second alternate protocol describes preparation of dot blots by spotting the samples onto the membrane by hand.
Synthesis and Purification of Oligonucleotides
Authors: Ellington Andrew , Pollard Jack D.
Published: June 2004

This unit provides strategies on 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 of DNA and RNA oligonucleotides.
Purification of Oligonucleotides Using Denaturing Polyacrylamide Gel Electrophoresis
Authors: Ellington Andrew , Pollard Jack D.
Published: June 2004

Cloning vectors derived from filamentous phage are extremely useful because they allow cloned DNA to be isolated as either single‐ or double‐stranded DNA. This unit contains protocols for preparing both forms of DNA and for characterizing inserts in M13‐derived vectors. A protocol is also presented for preparing single‐stranded DNA from plasmids using superinfection with helper phage. This method is advantageous because it allows cloned DNA to be maintained in the form of a plasmid while permitting single‐stranded DNA to be isolated for DNA sequencing.
Mapping by Multiple Endonuclease Digestions
Authors: Bloch Kenneth D.
Published: June 2004

In this unit, a protocol is provided in which the DNA of interest is cleaved with a variety of restriction endonucleases, either individually or in combination, and the resulting products are separated by agarose or acrylamide gel electrophoresis. By determining the sizes of DNA fragments produced by endonuclease cleavage, the restriction map is deduced progressively from simple situations where enzymes cleave the DNA once or twice to more complex situations where cleavage occurs more frequently.
Reagents and Radioisotopes Used to Manipulate Nucleic Acids
Authors: Struhl Kevin
Published: June 2004

This unit provides recipes and instructions for preparing many common and useful solutions that are used in nucleic acids research. Included are enzyme buffers, reaction conditions for many restriction enzymes, stock solutions of nucleoside triphosphates, as well as numerous procedures for isolating and quantifying the radioactivity of various radioisotopes.
Template‐Independent DNA Polymerases
Authors: Tabor Stanley
Published: June 2004

Terminal deoxynucleotidyl transferase, purified from calf thymus, catalyzes the incorporation of deoxynucleotides to the 3′‐hydroxyl termini of DNA accompanied by the release of inorganic phosphate. Reaction conditions are described in this unit in addition to some applications, including cloning DNA fragments, labeling the 3′ termini of DNA with 32P, incorporating nonradioactive tags onto the 3′ termini of DNA fragments, and synthesizing model polydeoxynucleotide homopolymers.
DNA‐Independent RNA Polymerases
Authors: Tabor Stanley
Published: June 2004

Poly(A) polyermase, purified from E. coli, catalyzes the incorporation of AMP residues onto the free 3′‐hydroxyl terminus of RNA, utilizing ATP as a precursor. This unit describes specific reaction conditions as well as some applications including labeling the 3′ ends of RNA with α32ATP for hybridization probes, and cloning RNA that lacks a poly(A) tail. The poly(A) tail is synthesized by poly(A) polyermase, and cDNA is synthesized from the resulting RNA using oligo(dT) as a primer.