bluuebunny/crossref_metadata_2025 huggingface dataset:
Traumatic Brain Injury
Authors: Auber Andrew E. , Belden Clifford
Published: June 2004

Magnetic Resonance (MR) imaging of the brain following head injury is used in two distinct clinical contexts, (1) acutely, within days of the injury, to evaluate an unexplained neurologic deficit or to obtain prognostic information, and (2) chronically, to assess the degree of brain injury and explain neurologic or neuropsychologic findings. In this unit, two basic protocols are presented, one for acute imaging and the other for chronic imaging. Advanced MR imaging sequences, such as MR spectroscopy (MRS) and diffusion‐weighted (DW) imaging can provide additional prognostic information in the acute setting and are also described. MR angiography (MRA) and direct vessel wall imaging techniques are mentioned briefly.
Multiple Sclerosis
Authors: Hallam Danial K.
Published: June 2004

Although a routine brain MR exam would suffice in many applications, specific protocols for multiple sclerosis have been developed to maximize its contribution. This unit presents one such protocol while recognizing the wide opportunity for variation depending on local clinical demand and personal preference.
Dementia
Authors: Hallam Danial K. , Salamon‐Muramaya Noriko
Published: June 2004

This unit presents specific protocols intended to maximize the contribution of MRI to the clinical evaluation of dementia. Each protocol has, as its foundation, a whole‐brain screen designed to evaluate for treatable causes of dementia. Reflecting the frequent overlap of vascular disease with dementing illness, the screening protocol also serves as an optimal evaluation of the patient with suspected vascular dementia. Additional sequences or modifications are then used to answer specific clinical questions. 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.
Quality Assurance for Clinical fMRI
Authors: Thulborn Keith R. , Davis Denise
Published: June 2004

The functional MRI (fMRI) procedure has several sources of variance that determine the success of the examination. These include the scanner, patient, and paradigm. As blood oxygenation level dependent (BOLD) contrast is a small effect, high signal‐to‐noise performance is mandatory. Because the preparation of a functional activation map requires averaging multiple images over time, the scanner must produce high temporal stability of the signal intensity. This unit presents the for achieving scanner stability. There are many determinants of such performance but not all possibilities need to be checked separately. An adequate approach has been to verify total system performance under the conditions of a functional MRI study on a phantom. This testing is done daily prior to patient studies.
Diffusion Tensor Imaging
Authors: Zhou Xiaohong Joe , Thulborn Keith R.
Published: June 2004

This unit provides step‐by‐step instructions on how to perform diffusion tensor imaging (DTI) in a clinical setting. A brief introduction on DTI techniques and current clinical applications is also presented. Additional technical details, practical considerations, and anticipated results are discussed in a commentary section.
Cranial Nerves VII To VIII
Authors: Evers Robert W. , Yousem David M.
Published: June 2004

Cranial nerves VII and VIII are easily visualized within cerebrospinal fluid (CSF) in most patients and are the nerves most commonly involved with neurogenic neoplasms. The reasons for performing contrast‐enhanced scans are explained in this unit, but revolve around pathologies other than schwannomas of the nerves.
Congenital Heart Disease
Authors: Woodard Pamela K. , Zheng Jie
Published: June 2004

Cardiac MRI plays a pivotal role in both anatomical and functional assessment of shunts, admixture lesions, transpositions, and the surgical correction of these lesions. This unit presents basic techniques for the evaluation of congenital heart disease. While sequence parameters described are meant to be as generic as possible, parameters are most appropriate for the Siemens 1.5 T Vision or Symphony, and may need to be altered for magnets of different field strengths and manufacturers.
MRI of the Liver
Authors: Fisher Laurie , Semelka Richard , Wilber Kathy
Published: June 2004

MRI provides comprehensive information on the full range of liver diseases, including congenital abnormalities, benign and malignant focal liver lesions, and diffuse liver disease. This unit presents a protocol that incorporates various types of T1‐and T2‐weighted sequences, including transverse and coronal data acquisition, and the routine use of intravenous gadolinium.
MRI of the Kidney
Authors: Semelka Richard , Fisher Laurie , Wilber Kathy
Published: June 2004

MRI 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

MRI 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 Pancreas
Authors: Semelka Richard , Fisher Laurie , Wilber Kathy
Published: June 2004

MRI provides comprehensive information on the full range of pancreatic diseases. This unit presents a protocol incorporating various types of sequences, including transverse and coronal data acquisitions, and with the routine use of intravenous gadolinium.
MRI of the Male Pelvis
Authors: Semelka Richard , Fisher Laurie , Wilber Kathy
Published: June 2004

MRI 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

In 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

The 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

At 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

In 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

The 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

This 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

Magnetic 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

This 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

This 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

The “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).
Media Preparation and Bacteriological Tools
Authors: Elbing Karen , Brent Roger
Published: June 2004

Recipes are provided in this unit for minimal liquid media, rich liquid media, solid media, top agar, and stab agar. Also included are descriptions and useful information about tools used with growth media such as inoculating loops, sterile toothpicks and spreaders.
Growth in Liquid Media
Authors: Elbing Karen , Brent Roger
Published: June 2004

The procedure for inoculating overnight (starter) cultures of E. coli from a single colony is described along with considerations for growing larger cultures. Also included are two methods for monitoring cell growth using a spectrophotometer or a hemacytometer.
Growth on Solid Media
Authors: Elbing Karen , Brent Roger
Published: June 2004

Detailed protocols are provided for titering and isolating bacterial colonies by serial dilutions, or alternatively by streaking or spreading a plate. Support protocols describe replica plating as well as methods for storing strains as agar stabs or frozen glycerol stocks.
Selected Topics from Classical Bacterial Genetics
Authors: Raleigh Elisabeth A. , Elbing Karen , Brent Roger
Published: June 2004

Current 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

An 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

Transformation 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

DNA 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

The 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

Most 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

Large 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

Capillary 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

The 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

Reverse 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

DNA 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

The 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

The 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

This 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

Sequence‐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

This 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

Specific 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 .
Identification of Newly Transcribed RNA
Authors: Greenberg Michael E. , Bender Timothy P.
Published: June 2004

Newly transcribed RNA can be identified using the nuclear runoff transcription assay. Isolated nuclei, free of membranes and cytoplasmic debris, are required for the assay. Cell lysis that does not allow the isolation of nuclei free of cell membranes and cytoplasmic material often results in poor incorporation of 32P‐labeled UTP into nascent transcripts. Although there is no way to predict what cell types present this problem, many adherent cell lines and lymphocytes isolated from murine spleen or thymus do. Interestingly, very few nonadherent cell lines have posed this problem. As described in this unit, isolating nuclei by detergent lysis of cells works well for many tissue culture cell lines but may not be appropriate for all cell lines and many tissues. Protocols for detergent lysis and Dounce homogenization or cell lysis in an isoosmotic solution and centrifugation through a sucrose cushion are described along with a for the preparation of the cDNA nitrocellulose filter strips that are used to detect the presence of specific transcripts in the nuclear runoff transcription assay.
Size Fractionation Using Sucrose Gradients
Authors: Weis John H. , Quertermous Thomas
Published: June 2004

DNA fragments migrate through a linear sucrose gradient at a rate that is dependent on their size. This procedure described in this unit provides good resolution for DNA fragments 5 to 60 kb in size, and sucrose gradients are also useful for purification of bacteriophage l vector arms. Partially digested genomic DNA can be fractionated for the production of cosmid or bacteriophage libraries and completely digested DNA can be fractionated for subgenomic DNA libraries. Protocols are provided in this unit for both partial and complete enzyme digestion of genomic DNA.
Production of a Complete cDNA Library
Authors: Klickstein Lloyd B.
Published: June 2004

A complete cDNA library is one that contains at least one cDNA clone representing each mRNA in the cell. A method is provided in this unit for calculating the required base of a cDNA library such that the library should contain at least one copy of every mRNA. Either a phage or plasmid vector can be used in constructing a complete cDNA library, and protocols are described for both. Procedures for evaluating these cDNA libraries are also provided along with a procedure for evaluating the quality of the λ arms used in constructing complete phage cDNA libraries.
Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries
Authors: Osoegawa Kazutoyo , de Jong Pieter J. , Frengen Eirik , Ioannou Panayiotis A.
Published: June 2004

Large‐insert genomic libraries are necessary for physical mapping of large chromosomal regions, for isolation of complete genes, and for use as intermediates in DNA sequencing of entire genomes. Construction of BAC and PAC libraries is detailed in the unit, including preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, dephosphorylation with alkaline phosphatase, and purification through pulsed‐field gel electrophoresis (PFGE). For the preparation of high‐molecular weight DNA for cloning, procedures for embedding total genomic DNA from lymphocytes or animal tissue cells are also provided. Other protocols detail partial digestion of genomic DNA with MboI or with a combination of EcoRI endonuclease and EcoRI methylase (including methods for optimizing the extent of digestion), and subsequent size fractionation by preparative PFGE. Finally, the isolation of BAC and PAC plasmid DNA for analyzing clones is also presented.
Plating and Transferring Cosmid and Plasmid Libraries
Authors: Weis John H.
Published: June 2004

There are two commonly used protocols for the screening of recombinant bacteria with hybridization probes. One method involves the spreading of bacteria on the surface of agar using a sterile spreader. A nitrocellulose membrane filter is then placed on top of the colonies and most of each colony is transferred to the filter. This method works well when relatively small numbers of positive colonies are being selected (up to several thousand). A second method, described in this unit, employs a matrix of some type (here nitrocellulose filters are used) upon which bacteria can be plated and grown when the filter is placed on top of a nutrient agar surface. Once the plated bacteria have grown into visible colonies, the filters can be used for replica plating and in situ hybridization analysis.
Using DNA Fragments as Probes
Authors: Strauss William M.
Published: June 2004

All hybridization methods depend upon the ability of denatured DNA to reanneal when complementary strands are present in an environment near but below their Tm (melting temperature). A detailed procedure is described in which bacteriophage plaques or bacterial colonies bound to a filter membrane are detected by hybridization with a radioactive probe. Hybridization proceeds on prewet filters placed in a sealable plastic bag. After hybridization the filters are removed from the sealed bag, excess probe is washed off, and the filters are autoradiographed to identify the clones that have hybridized with the probe. An differs mainly in that formamide is not used in the hybridization solution.
Purification of Cosmid and Plasmid Clones
Authors: Weis John H.
Published: June 2004

Cosmid‐ or plasmid‐bearing colonies that are identified by hybridization are purified by spreading the cosmids or plasmids on an agar plate and repeating the colony hybridization.
Immunoscreening of Fusion Proteins Produced in Lambda Plaques
Authors: St. John Thomas P.
Published: June 2004

Screening large numbers of plaques containing particular proteins is accomplished by techniques that are analogous to those described for screening with radioactive DNA probes. However, in the basic protocol described here, the plaques are screened with antibodies specific to the desired proteins. An alternate protocol provides a method for increasing the amount of recombinant protein in each plaque by inducing expression from the lac promoter that directs its expression.
DNA Sequencing by the Dideoxy Method
Authors: Slatko Barton E. , Albright Lisa M. , Tabor Stanley , Ju Jingyue
Published: June 2004

In the basic dideoxy sequencing reaction, an oligonucleotide primer is annealed to a single‐stranded DNA template and extended by DNA polymerase in the presence of four deoxyribonucleoside triphosphates (dNTPs), one of which is 35S‐labeled. The reaction also contains one of four dideoxyribonucleoside triphosphates (ddNTPs), which terminate elongation when incorporated into the growing DNA chain. After completion of the sequencing reactions, the products are subjected to electrophoresis on a high‐resolution denaturing polyacrylamide gel and then autoradiographed to visualize the DNA sequence. Three variations of the dideoxy sequencing procedure are currently in use and are presented in this unit. In the “labeling/termination“ procedure, primer chains are initially extended and labeled in the absence of terminating ddNTPs, whereas in the traditional “Sanger” procedure, labeling and termination of primer chains occur in a single step. A recent variation of the dideoxy sequencing method is thermal cycle sequencing in which the reaction mixture, containing template DNA, primer, thermostable DNA polymerase, dNTPs, and ddNTPs, is subjected to repeated rounds of denaturation, annealing, and elongation steps. The resulting linear amplification of the sequencing products allows much less template DNA to be used and eliminates independent primer annealing and template denaturation steps, which are required for the labeling/termination or Sanger procedures. The use of automated fluorescent sequencers ofr four‐color dideoxy DNA sequencing is also described in detail.
Mutagenesis with Degenerate Oligonucleotides: Creating Numerous Mutations in a Small DNA Sequence
Authors: Hill David E.
Published: June 2004

The procedure described in this unit is an efficient method for using the products of a single oligonucleotide synthesis to create numerous mutations in a small region of DNA (20 to 80 nucleotides). Mutant oligonucleotides are generated during the organic synthesis reaction by including low concentrations of the three non‐wild‐type nucleotide precursors with the wild‐type precursor at each step of the synthesis. The product of such a DNA synthesis is a degenerate oligonucleotide, i.e., a complex mixture of related molecules, each of which has a defined probability of being altered from the wild‐type sequence. The frequencies and types of zero, single, double, and higher order mutations can be set simply by choosing the appropriate amounts of non‐wild‐type precursors at each step of nucleotide addition during the synthesis.
Random Mutagenesis by PCR
Authors: Wilson David S. , Keefe Anthony D.
Published: June 2004

Error‐prone PCR (EP‐PCR) is the method of choice for introducing random mutations into a defined segment of DNA that is too long to be chemically synthesized as a degenerate sequence. Using EP‐PCR, the 5′ and 3′ boundaries of the mutated region may be defined by the choice of PCR primers. Accordingly, it is possible to mutagenize an entire gene or merely a segment of a gene. The average number of mutations per DNA fragment can be controlled as a function of the number of EP‐PCR doublings performed. The EP‐PCR technique described here is for a 400‐bp sequence, and an is for a library. EP‐PCR takes advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the addition of Mn2+, increasing the Mg2+ concentration, and using unequal dNTP concentrations.
Directed Mutagenesis Using the Polymerase Chain Reaction
Authors: Cormack Brendan
Published: June 2004

This unit contains two basic protocols for introducing base changes into specific DNA sequences. The first describes the incorporation of a restriction site and the second details the generation of specific point mutations. An describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.
Direct Analysis of RNA after Transfection
Authors: Kingston Robert E.
Published: June 2004

It is possible to transfect mammalian cells with a gene of interest and directly detect the RNA made from that gene 24 to 48 hr later. The ability to analyze the RNA directly allows mutation and functional analysis of an intact gene, as the promoter does not have to be subcloned. This technique is also essential when fusion genes are used. In order to be sure that the level of the reporter protein produced by a fusion gene provides an accurate measure of appropriately initiated RNA from the promoter under study, it is necessary to determine the amount and 5′ end of the fusion message. An investigator who verifies this using direct RNA analysis can proceed with some confidence that the level of the reporter protein is a measure of promoter activity. The difficulty in directly analyzing RNA is that the sensitivity of detection is at the limits of present technology. Consequently, the transfection protocol, RNA preparation, and RNA analysis techniques all have to be working near optimum in order for an experiment to work. This overview discusses parameters that must be considered when directly analyzing RNA after a transfection: transfection efficiency, the method of preparation of the RNA, the method of analysis of RNA, and strength of the promoter.
Overview of the Retrovirus Transduction System
Authors: Cepko Constance , Pear Warren
Published: June 2004

A retrovirus vector is an infectious virus used to introduce a nonviral gene into mitotic cells in vivo or in vitro. The efficient and precise integration machinery of naturally occurring retroviruses is utilized to produce either a single copy or a few copies of the viral genome stably integrated into a host chromosome. This unit presents an overview of the retrovirus life cycle and a description of vector designs and packaging cell lines.
Preparation of a Specific Retrovirus Producer Cell Line
Authors: Cepko Constance
Published: June 2004

Establishing a cell line that produces high levels of a specific retrovirus construct involves several steps. First, the retroviral construct must be stably introduced into an appropriate packaging cell line. This can be accomplished either directly by transfection or by transfection to produce a transient virus stock followed by cross‐infection of this stock into a separate packaging line; procedures for both approaches are described in this unit. After stable lines are produced, they must be characterized to identify lines that produce high titers of virus with an appropriate structure. A drug selection protocol (the most common method for determining virus titer) is provided along with a sample calculation of BAG virus titer. Sometimes, a short and direct method of estimating virus titer is available when the virus encodes a histochemically detectable gene such as lacZ. For this case, an Xgal staining protocol is described. Similarly, a protocol is provided to estimate the levels of virus being produced by a given cell line by quantitating any specific product of the virus (e.g., RNA) present within the producer line.
Transient Transfection Methods for Preparation of High‐Titer Retroviral Supernatants
Authors: Pear Warren
Published: June 2004

Generation of high‐titer retrovirus by transient production not only is less laborious than production of stable retroviral producer cell lines, but also has allowed the production of high‐titer retroviral supernatants from cDNAs that cannot be achieved by stable producer cell lines. Transient transfection has also increased the versatility of retrovirus‐mediated gene transfer to include the rapid testing of different constructs, viral pseudotyping, and construction of retroviral cDNA libraries. Systems based on human 293 cells, an adenovirus‐transformed human embryonic kidney cell line have produced the highest retroviral titers and are the most widely used. This unit describes methods for optimizing retroviral production from the 293‐based systems and for growing and freezing 293 cells. Methods are included for pseudotyping the virus with VSV G protein by sequential transfection or cotransfection. Virus produced by transiently transfected cells can be used to infect cells. Protocols are provided for infection of adherent cells either directly with retroviral supernatant or by spin infection. In addition, procedures are included for infection of nonadherent cells by addition of retrovirus supernatant, cocultivation with producer cells, or spin infection. These infection methods are also applicable to retrovirus produced by any of the stable producer cell lines.
Large‐Scale Preparation and Concentration of Retrovirus Stocks
Authors: Cepko Constance
Published: June 2004

For some applications, such as infection of cells in vivo, it is necessary to concentrate retrovirus stocks in order to increase their titer. Because viruses are macromolecular structures, they can be concentrated fairly easily by a relatively short centrifugation step. This unit provides protocols in which the viral particles are either pelleted or centrifuged onto a sucrose density step gradient. An alternate protocol details how virions can be precipitated using polyethylene glycol or ammonium sulfate, and the resulting precipitate collected by centrifugation. After resuspension of the precipitates, the virions can be used directly, or further purified either by sedimentation onto sucrose density gradients, or by column filtration as described here. Perhaps most simply of all, an alternate procedure describes how small volumes of virus stock can be concentrated by centrifugation through a filter that allows only small molecules to pass.
Retrovirus Infection of Cells In Vitro and In Vivo
Authors: Cepko Constance , Pear Warren
Published: June 2004

There are many applications in which retrovirus vectors are used as transduction agents. In some cases, the vector carries a gene that one wishes to express in a target cell in order to study the function of that gene. In other cases, the virus is used to introduce a histochemical marker gene into cells in order to follow their fate. Retrovirus vectors can also be used in a variety of cells type to investigate regulatory sequences in which a reporter gene and regulatory sequences are carried by the vector and to immortalize or transform primary cells by transduction of oncogenes. For each application, the infection protocol may vary and must often be optimized. Guidelines for infection of cells in some typical in vivo and in vitro experiments are presented in this overview.
Human Somatic Cell Gene Targeting
Authors: Waldman Todd , Lee Carolyn , Nishanian Tagvor G. , Kim Jung‐Sik
Published: June 2004

Human somatic cell gene targeting provides a powerful tool to scientists studying gene function in cultured human cells. This technology allows scientists to knock out genes in human somatic cells in a fashion analogous to the creation of knockout mice. Human somatic cell gene targeting brings the power of genetics to the study of human genes in human cells by making it possible to compare cells or individuals that are genetically identical except for a single, well‐defined mutation in an endogenous gene. These modified cells can be studied both in vitro and in vivo. This unit presents protocols for human somatic cell gene targeting.