bluuebunny/crossref_metadata_2025_split huggingface dataset:
MRI of the Kidney
Authors: Semelka Richard , Fisher Laurie , Wilber Kathy 
Published: June 2004

AbstractMRI provides comprehensive information on the full range of kidney diseases, including benign and malignant processes and diffuse renal parenchymal disease. This unit presents a set protocols incorporating various types of sequences including transverse, coronal and sagittal data acquisition, and the routine use of intravenous gadolinium.
MRI of the Adrenal Glands
Authors: Wilber Kathy , Semelka Richard , Fisher Laurie 
Published: June 2004

AbstractMRI is an effective method to evaluate adrenal pathology. This unit presents a protocol incorporating various types of T1‐ and T2‐weighted sequences, including the use of in‐phase and out‐of‐phase gradient echo techniques and the routine use of intravenous gadolinium.
MRI of the Male Pelvis
Authors: Semelka Richard , Fisher Laurie , Wilber Kathy 
Published: June 2004

AbstractMRI provides comprehensive information on many male pelvic diseases, such as prostate cancer and bladder cancer as well as lymph nodes and bone metastases. This unit presents a protocol incorporating T1‐ and T2‐weighted sequences including transverse and sagittal data acquisitions, and the routine use of intravenous gadolinium.
MRI of Breast Lesions
Authors: Kaiser Werner A. 
Published: June 2004

AbstractIn Magnetic Resonance Mammography (MRM) high spatial as well as temporal resolution is of utmost importance for differentiating between malignant and benign lesions. Therefore, a so‐called dynamic technique (i.e., the repetitive imaging of the same slices before and in short time intervals after the injection of contrast medium) is essential to detect the differences in initial enhancements between malignant and benign lesions which are reflected by the tumorangiogenetic vascular network of malignant lesions. This unit presents a basic protocol and several alternate protocols for dynamic MRM.
Contrast‐Enhanced Renal MRA
Authors: Shetty Anil N. , Bis Kostaki G. 
Published: June 2004

AbstractThe rapid growth of magnetic resonance imaging systems with enhanced gradient systems together with improved pulse sequences has improved the ability to image blood vessels with a spatial and temporal resolution similar to conventional X‐ray angiography. With patients who cannot undergo X‐ray angiography because they are contraindicated for iodinated contrast agents (having a creatinine level > 2.0), MRA (magnetic resonance angiography) has proven to be the modality of choice. Since the first demonstration of such contrast‐enhanced studies in the abdominal aorta, there have been continual improvements in methods due to improved hardware/software capabilities. This unit presents the MR protocols to image vascular morphology using contrast‐enhanced 3‐D‐MRA techniques. The pulse sequences described herein are based on the authors' experience with a Siemens 1.5 T Vision and 1.5 T Sonata scanners, but are expected to be equally applicable to machines from other manufacturers.
Spin Behavior in the Rotating Reference Frame
Authors: Cheng Yu‐Chung Norman , Haacke E. Mark 
Published: June 2004

AbstractAt any given point in time, the interaction of a classical magnetic moment with an external magnetic field is equivalent to an instantaneous rotation of the moment about the field. For a static field, the rotation is a constant precession about the field itself. This unit discusses the effect of adding a radiofrequency (RF) field perpendicular to a much larger constant field. The interest in the additional field stems from the need to generate a transverse component of the magnetization.
Fundamental Properties of Magnetization
Authors: Cheng Yu‐Chung Norman , Haacke E. Mark 
Published: June 2004

AbstractIn MR imaging the detected signal is proportional to the magnetization of an ensemble of spins (a spin isochromat). Thus, one can simply replace the magnetic moment with bulk magnetization in many equations such as the Bloch equations. The beauty is that the physics concepts behind energy, forces, and the equations of motion do not change, whether one considers a single spin or a set of spins. In this unit, the concept of bulk magnetization and quantized spin angular momentum are introduced. The dependence of the magnetization on spin density.
Signal Detection
Authors: Cheng Yu‐Chung Norman , Haacke E. Mark 
Published: June 2004

AbstractThe bulk magnetization represents the equilibrium value of the magnetization when all spins have aligned themselves parallel to the main field. When this magnetization is tipped into the transverse plane, it precesses about the main field. This precession generates a changing flux through the face of an appropriately placed coil inducing an electromotive force (emf) in the coil. The measured signal is proportional to the induced emf. This unit discusses the basic physics behind detecting MR signal. The process of calculating induced emf based on Faradays law of induction is explained.
Spin Density, T1, T2, T2* Relaxation and Bloch Equations
Authors: An Hongyu , Lin Weili 
Published: June 2004

AbstractThis unit discusses the four primary sources of signal variation in a standard MRI sequence. T1 and T2/T2* relaxation reveal the time dependence of the longitudinal and transverse component of the magnetization, respectively. When imaging is performed, the effective spin density is considered as the number of spins per voxel. The variation of signal with voxel size is discussed. Modification of Bloch equations that govern the motion of the magnetization is discussed for all four cases.
One‐Dimensional Fourier Imaging and k‐Space
Authors: Lin Weili 
Published: June 2004

AbstractMagnetic resonance imaging offers the possibility to obtain spatially resolved anatomical information. This is accomplished by taking advantage of the Larmor relationship, which dictates that the frequency of the spins depends on the local magnetic field. This unit discusses the one dimensional Fourier imaging based on this relation. The one‐to‐one mapping of the signal from a given frequency to a given spatial location is explained. The image reconstruction based on well‐known Fourier transform reconstruction method is described in detail. The Fourier transform takes the MR signal as acquired in the time domain (usually referred to as the k‐space domain) and converts it to the frequency domain where 1‐D spatially resolved information can be obtained.
Phase Dependence on Motion
Authors: Cheng Yu‐Chung Norman , Haacke E. Mark 
Published: June 2004

AbstractThis unit introduces the sources of variable phase in the presence of motion. The sources of these phase changes can come from the read gradient, from the slice select gradient and from the phase encoding gradient. Motion during the gradient moves the spin from one location to another and, therefore, changes the effective field it sees. This field change leads to a frequency change in time during either the dephasing or rephasing gradient or both. As a result, the spins do not refocus at the expected time.
Motion Compensation Along All Three Different Directions
Authors: Norman Cheng Yu‐Chung , Haacke E. Mark 
Published: June 2004

AbstractThis unit addresses how one can deal with phase perturbations that arise due to the presence of motion. Techniques to reduce or remove their associated artifacts are explained in detail. Artifacts due to phase changes that come from the read gradient, from the slice select gradient and from the phase encoding gradient are also discussed.
Magnetic Resonance Angiography
Authors: Norman Cheng Yu‐Chung , Haacke E. Mark 
Published: June 2004

AbstractThe “flow compensation” of moving spins is necessary to visualize blood in MR imaging. This unit discusses the effects of blood flow in detail, particularly in regard to obtaining an accurate measurement of the blood vessel lumen (the interior dimensions of the vessel). The imaging of blood vessels (arteries and veins) is often referred to as magnetic resonance angiography (MRA).
Selected Topics from Classical Bacterial Genetics
Authors: Raleigh Elisabeth A. , Elbing Karen , Brent Roger 
Published: June 2004

AbstractCurrent cloning technology exploits many facts learned from classical bacterial genetics. This unit covers those that are critical to understanding the techniques described in this book. Topics include antibiotics, the LAC operon, the F factor, nonsense suppressors, genetic markers, genotype and phenotype, DNA restriction, modification and methylation and recombination.
Introduction to Plasmid Biology
Authors: Feinbaum Rhonda 
Published: June 2004

AbstractAn array of issues pertaining to plasmid biology are presented in this unit. Topics include replicators, an explanation of the mechanism of replication and copy‐number control, plasmid incompatibility, selectable markers, cloning sites, and factors to consider when choosing a plasmid vector, whether it be for production of ssDNA, cloning of large inserts, or expression of large quantities of recombinant proteins. Considerations are discussed for the use of plasmids in yeast, cultured mammalian cells and non‐E. coli bacteria. Finally, maps of many common useful plasmids are presented. This unit has recently been updated and extensively revised to include some of the latest developments in plasmid construction and experimental use.
Introduction of Plasmid DNA into Cells
Authors: Seidman Christine E. , Struhl Kevin , Sheen Jen , Jessen Timm 
Published: June 2004

AbstractTransformation of E. coli can be achieved using any of the four protocols in this unit. The first method using calcium chloride gives good transformation efficiencies, is simple to complete, requires no special equipment, and allows storage of competent cells. The alternate one‐step method is considerably faster and also gives good transformation efficiencies (although they are somewhat lower). If considerably higher transformation efficiencies are needed, the third method using electroporation is simple, fast, and reliable. As in the calcium chloride protocol, prepared cells can be stored. The final method described is an adaptation of the electroporation protocol that allows direct transfer of vector DNA from yeast into E. coli.
Preparing Lambda DNA from Phage Lysates
Authors: Lech Karen , Reddy K.J. , Sherman L.A. 
Published: June 2004

AbstractDNA extracted from lambda‐derived vectors is typically subcloned into plasmid or filamentous phage vectors. The first two protocols describe methods for isolating phage DNA from large‐ and medium‐scale liquid lysates. These two methods use either density‐gradient centrifugation or ion‐exchange chromatography to purify the phage particles. The third protocol describes a rapid procedure for isolating phage DNA, suitable for small‐scale liquid lysates.
Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination
Authors: Thomason Lynn , Court Donald L. , Bubunenko Mikail , Costantino Nina , Wilson Helen , Oppenheim Amos 
Published: June 2004

AbstractThe bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage‐encoded recombination functions efficiently recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with double‐stranded DNA (dsDNA) or single‐stranded DNA (ssDNA). Support protocols describe a two‐step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a coelectroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.
Preparation of Genomic DNA from Bacteria
Authors: Wilson Kate 
Published: June 2004

AbstractMost protocols for the preparation of bacterial genomic DNA consist of lysis, followed by incubation with a nonspecific protease and a series of extractions prior to precipitation of the nucleic acids. Such procedures effectively remove contaminating proteins, but are not effective in removing exopolysaccharides which can interfere with the activity of enzymes such as restriction endonucleases and ligases. In this unit, however, the protease incubation is followed by a CTAB extraction whereby CTAB complexes both with polysaccharides and with residual protein, effectively removing both in the subsequent emulsification and extraction. This procedure is effective in producing digestible chromosomal DNA from a variety of gram‐negative bacteria, all of which normally produce large amounts of polysaccharides. If large amounts of exceptionally clean DNA are required, the procedure can be scaled up and the DNA purified on a CsCl gradient, as described in the alternate protocol.
Separation of Small DNA Fragments by Conventional Gel Electrophoresis
Authors: Chory Joanne , Pollard Jack D. 
Published: June 2004

AbstractLarge amounts of small (<1000‐bp) DNA fragments can be separated by conventional electrophoretic means. The purified fragments can then be used for cloning, sequencing, and labeling. In this unit, the techniques of DNA separation via both nondenaturing polyacrylamide and sieving agarose electrophoresis are discussed. Methods are detailed for the pouring and electrophoresis of nondenaturing polyacrylamide gels, followed by elution of the labeled or unlabeled separated DNA fragments from the gels by either passive diffusion or electroelution. A method is also provided for the use of sieving agarose, a specially treated type of agarose designed to be used at high concentrations. Poured and run like conventional agarose gels, this matrix can resolve small DNA fragments much like a nondenaturing polyacrylamide gel.
Capillary Electrophoresis of DNA
Authors: Smith Alan , Nelson Robert J. 
Published: June 2004

AbstractCapillary electrophoresis (CE) is an alternative to conventional slab gel electrophoresis for the separation of DNA fragments. CE offers a number of advantages over slab gel separations in terms of speed, resolution, sensitivity, and data handling. Separation times are generally only a few minutes and the DNA is detected either by UV absorption or by fluorescent labeling. The quantity of DNA required for the separation is in the nanogram range. Single‐base resolution can be obtained on fragments up to several hundred base pairs. In the presence of appropriate standards, fragments can be accurately sized, based on relative electrophoretic mobility. A protocol for the analysis of synthetic oligonucleotides in a flowable matrix is described in this unit as an example of an application of CE. Another area of growing interest is the ability to analyze low levels of PCR products in biological fluids, and a protocol is presented for this purpose.
Hybridization Analysis of DNA Blots
Authors: Brown Terry 
Published: June 2004

AbstractThe principle of hybridization analysis is that a single‐stranded DNA or RNA molecule of defined sequence (the “probe”) can base‐pair to a second DNA or RNA molecule that contains a complementary sequence (the “target”) that has been immobilized on a nitrocellulose and nylon (uncharged and charged) membrane support. The approach taken in this unit is to present as the basic protocol an unsophisticated procedure for hybridization analysis with a radiolabeled DNA probe. The alternate protocol describes a similar method for probing DNA blots with a radiolabeled RNA probe. A support protocol for stripping blots for reprobing is also provided. The commentary describes modifications, including changes to prehybridization, hybridization, and wash solution formulations, and alterations to incubation times and conditions, the latter including a discussion of the wash conditions compatible with different degrees of stringency.
RNA‐Dependent DNA Polymerases
Authors: Tabor Stanley 
Published: June 2004

AbstractReverse transcriptases are derived from retroviruses which use them to make DNA copies of their RNA genomes. They are multifunctional enzymes, but they are mainly used as RNA‐directed DNA polymerases. Specifically, deoxyoligonucleotides (either oligo(dT) polymers, a collection of random DNA sequences, or a specific sequence) are used as primersfor extension on RNA (usually messenger RNA) templates. The DNA synthesized from the RNA templa te is referred to as complementary DNA (cDNA). This unit describes appropriate reaction conditions along with applications such as synthesizing cDNA for insertion into bacterial cloning vectors, filling in and labeling the 3′termini of DNA fragments with 5' protruding ends, and its use for DNA sequencing in place of Klenow fragment.
DNA Ligases
Authors: Tabor Stanley 
Published: June 2004

AbstractDNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5' phosphate and a 3'‐hydroxyl terminus in duplex DNA. This activity can repair single‐stranded nicks in duplex DNA and join duplex DNA restriction fragments having either blunt ends or homologous cohesive ends. Two ligases are used for nucleic acid research and their reaction conditions and applications are described in this unit: E. coli ligase and T4 ligase. These enzymes differ in two important properties. One is the source of energy: T4 ligase uses ATP, while E. coli ligase uses NAD. Another important difference is their ability to ligate blunt ends; under normal reaction conditions, only T4 DNA ligase will ligate blunt ends.
Subcloning of DNA Fragments
Authors: Struhl Kevin 
Published: June 2004

AbstractThe essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of insert sequences into plasmid or bacteriophage cloning vectors. The insert sequences can derive from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA segments (in which case, the procedure is termed subcloning). Alternatively, insert DNAs can be created directly by DNAsynthesis. This unit provides protocols for the subcloning of DNA fragments and ligation of DNA fragments in gels.
Preparation of Cytoplasmic RNA from Tissue Culture Cells
Authors: Gilman Michael 
Published: June 2004

AbstractThe protocol described in this unit is a fast and streamlined procedure for preparing total cytoplasmic RNA from many cultures simultaneously for nuclease protection analysis. It is scaled for small cultures‐‐1 to 2 dishes of adherent cells or 10 to 20 ml of a suspension culture. The procedure works well for many cell types. If full‐length RNA is required, ribonuclease inhibitors should be added to the lysis buffer (as described in this unit) or the guanidinium isothiocyanate method should be used. Finally, if RNA is isolated from transiently transfected cells, a is provided for the treatment of RNA with deoxyribonuclease to remove transfected DNA. This modification is especially critical if the RNA is to be assayed by nuclease protection using uniformly labeled probes.
S1 Analysis of Messenger RNA Using Single‐Stranded DNA Probes
Authors: Greene John M. , Struhl Kevin 
Published: June 2004

AbstractThis method takes advantage of the ability of oligonucleotides to be efficiently labeled to a high specific activity at the 5' end through the use of kinase. The oligonucleotide is hybridized to a specific single‐stranded template containing the complementary sequence to the oligonucleotide, and this hybrid is extended through the use of the Klenow fragment of E. coli DNA polymerase I. The mixture is cut with a restriction enzyme to give the probe a defined 3' end, and the probe is isolated on an alkaline agarose gel. Before using this protocol it is first helpful to have an M13 clone. If this is unavailable, a double‐stranded plasmid clone of the region to be studied may be used, as described in an alternate protocol. Another alternate protocol describes the use of long oligonucleotides as probes for S1 analysis (useful for rapid and easy quantitation of the level of mRNA produced from a characterized promoter). For the mapping of the 5' end of an RNA species, hybridization of the probe to RNA is then carried out. S1 nuclease is added to digest all of the unhybridized portion of the probe. Electrophoresis of the hybrid on a denaturing polyacrylamide gel allows a determination of the length of the remaining DNA fragment. This length equals the distance between the 5' end of the probe to the 5' end of the RNA, defining the transcriptional start site to the nucleotide. By performing the hybridization reaction in vast probe excess, quantitation of the relative amounts of RNA can be estimated between samples.
Ribonuclease Protection Assay
Authors: Gilman Michael 
Published: June 2004

AbstractSequence‐specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved with a restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance. The RNA is purified by removal of the DNA template, protein, and the unincorporated label. Alternatively, the probe is purified by gel electrophoresis, as described in a support protocol. The probe RNA is hybridized to sample RNAs and the hybridization reactions are treated with ribonuclease to remove free probe, leaving intact fragments of probe annealed to homologous sequences in the sample RNA. These fragments are analyzed by electrophoresis on a sequencing gel and the presence of the target mRNA is revealed by the appearance of an appropriately sized fragment of the probe.
Primer Extension
Authors: Triezenberg Steven J. 
Published: June 2004

AbstractThis protocol can be used to map the 5' terminus of an RNA and to quantitate the amount of a given RNA by extending a primer using reverse transcriptase. The primer is an oligonucleotide (or restriction fragment) that is complementary to a portion of the RNA of interest. The primer is end‐labeled, hybridized to the RNA, and extended by reverse transcriptase using unlabeled deoxynucleotides to form a single‐stranded DNA complementary to the template RNA. The resultant DNA is analyzed on a sequencing gel. The length of the extended primer maps the position of the 5' end of the RNA, and the yield of primer extension product reflects the abundance of the RNA.
Analysis of RNA by Northern and Slot Blot Hybridization
Authors: Brown Terry , Mackey Karol 
Published: June 2004

AbstractSpecific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting); unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Northern blotting differs from Southern blotting largely in the initial gel fractionation step. Because they are single‐stranded, most RNAs are able to form secondary structures by intramolecular base pairing and must therefore be electrophoresed under denaturing conditions if good separations are to be obtained. Denaturation is achieved either by adding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxal and dimethyl sulfoxide (DMSO) prior to loading. The describes blotting and hybridization of RNA fractionated in an agarose‐formaldehyde gel. Alternate protocols describe the glyoxal/DMSO method for denaturing gel electrophoresis and slot‐blot hybridization of RNA samples. Stripping hybridization probes from blots can be done under three different sets of conditions; these methods are outlined in a .