bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis and Purification of Oligonucleotides
Authors: Ellington Andrew , Pollard Jack D. 
Published: June 2004

AbstractThis 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

AbstractCloning 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

AbstractIn 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

AbstractThis 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

AbstractTerminal 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

AbstractPoly(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.
Phosphatases and Kinases
Authors: Tabor Stanley 
Published: June 2004

AbstractThe reaction conditions and applications of two phosphatases and one kinase are described in this unit. Bacterial alkaline phosphatase (BAP) from E. coli and calf intestine phosphatase (CIP) from veal are commonly used in nucleic acid research. Both enzymes catalyze the hydrolysis of 5′‐phosphate residues from DNA, RNA, and ribo‐ and deoxyribonucleoside triphosphates. The dephosphorylated products possess 5′‐hydroxyl termini which can subsequently be radioactively labeled using [γ‐32P]ATP and T4 polynucleotide kinase. T4 polynucleotide kinase has 3 activities: the forward reaction efficiently catalyzes the transfer of the terminal (γ) phosphate of ATP to the 5′‐hydroxyl termini of DNA and RNA. The exchange reaction catalyzes the exchange of 5'‐terminal phosphates. Lastly, T4 polynucleotide kinase is a 3′ phosphatase.
Ribonucleases
Authors: Struhl Kevin 
Published: June 2004

AbstractRibonucleases (RNases) with different sequence specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. One very common application for RNase A is presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. Two other commonly used RNases, RNase H and RNase T1, are also described. In addition, many commercially available RNases are sequence‐specific endoribonucleases, a property has been used for enzymatic sequencing of RNA.
Labeling and Colorimetric Detection of Nonisotopic Probes
Authors: Boyle Ann , Perry‐O'Keefe Heather 
Published: June 2004

AbstractAlthough a number of different nonisotopic labels have been described in the literature, biotin and digoxigenin are used most frequently and are commercially available. Either label can be easily incorporated into DNA probes and be detected colorimetrically; a number of fluorochromes, as well as alkaline phosphatase and horseradish peroxidase (which produce colored precipitates) are available directly conjugated to anti‐digoxigenin antibodies and to avidin. Chemiluminescent detection methods and indirect immunofluorescent techniques also provide sensitive alternatives for many molecular biology applications. This unit presents two protocols for incorporating biotinylated nucleotides into DNA probes by nick translation and random‐primed synthesis. A describes colorimetric detection of the probes, which also serves to check the extent of nucleotide incorporation. An describes adaptations of the basic protocols for incorporation of digoxigenin‐modified nucleotides.
Guanidine Methods for Total RNA Preparation
Authors: Kingston Robert E. , Chomczynski Piotr , Sacchi Nicoletta 
Published: June 2004

AbstractThree different methods for RNA preparation using guanidine are presented in this unit ‐‐ a single‐step isolation method employing liquid‐phase separation to selectively extract total RNA from tissues and cultured cells and two methods that rely on a CsCl step gradient to isolate total RNA.
Preparation of Bacterial RNA
Authors: Reddy K.J. , Gilman Michael 
Published: June 2004

AbstractProcedures for isolating RNA from bacteria involve disruption of the cells, followed by steps to separate the RNA from contaminating DNA and protein. Lysis strategies differ in the protocols presented in this unit, including chemical degradation of Gram‐negative cell walls using sucrose/detergent or lysozyme, and sonication to break open Gram‐positive cell walls. Combinations of enzymatic degradation, organic extraction, and alcohol or salt precipitation are employed in the procedures to isolate the RNA from other cellular components, and various inhibitors of ribonuclease activity (diethylpyrocarbonate, vanadyl‐ribonucleoside complex, and aurintricarboxylic acid) are described. If extremely high‐quality RNA is required (e.g., for gene expression studies), instructions are provided for CsCl step‐gradient centrifugation to remove all traces of contaminating DNA.
Preparation of Poly(A)+RNA
Authors: Kingston Robert E. 
Published: June 2004

AbstractMost messenger RNAs contain a poly(A) tail, while structural RNAs do not. Poly(A) selection therefore enriches for messenger RNA. The technique has proved essential for construction of cDNA libraries. It is also useful when analyzing the structure of low‐abundance mRNAs. Removing the ribosomal and tRNAs from a preparation increases the amount of RNA that can be clearly analyzed by S1 analysis, for example, thus allowing detection of a low level message. This protocol separates poly(A)+ RNA from the remainder of total RNA, which is largely rRNA and tRNA. Total RNA is denatured to expose the poly(A) (polyadenylated) tails. Poly(A)‐containing RNA is then bound to oligo(dT) cellulose, with the remainder of the RNA washing through. The poly(A)+ RNA is eluted by removing salt from the solution, thus destabilizing the dT:rA hybrid. The column can then be repeated to remove contaminating poly(A)− RNA.
cDNA Libraries
Authors: Moore David D. 
Published: June 2004

AbstractThis unit provides a basic description of issues that require careful consideration before undertaking construction of cDNA libraries. Topics include: generating a double‐stranded DNA copy of the mRNA, the quality of the mRNA, the relative abundance of the clone of interest, the choice of screening method, the size of the library that will be necessary to include the clone of interest, and choices of linkers or adaptors for insertion into the vector.
Size Fractionation Using Agarose Gels
Authors: Quertermous Thomas 
Published: June 2004

AbstractIn the , digested genomic DNA is size fractionated on a slab agarose gel, and the appropriate region of the gel is defined by Southern blot analysis (for subgenomic libraries) or by size (for genomic libraries) and the DNA is eluted. An describes electrophoresis using a Bull's‐eye apparatus. Digested genomic DNA is loaded onto a large preparative circular agarose gel in this apparatus, the DNA fragments are electrophoresed toward the center of the gel and then eluted. Fragments leaving the gel are pooled and constitute a fraction. Fractions containing the gene of interest are identified by Southern blotting of a small aliquot of alternate fractions. This procedure allows the purification of large amounts of size‐fractionated DNA that is particularly well suited for genomic library construction and normally allows creation of large numbers of recombinant clones.
Ligation of Linkers or Adapters to Double‐Stranded cDNA
Authors: Klickstein Lloyd B. , Neve Rachael L. 
Published: June 2004

AbstractThe describes the strategy of producing a genomic DNA library. A number of small‐scale ligations are performed using a set amount of vector and varying amounts of insert. Test ligations are transformed into bacteria (plasmid vectors) or packaged and plated on host bacteria (λ and cosmid vectors). The number of clones in the different ligations is compared, and the optimum ratio of vector to insert is indicated by the ligation with the most recombinant clones. A large‐scale ligation is then set up using this optimum ratio. This protocol employs a bacteriophage vector; however, cosmid or plasmid vectors can be used with minor modifications.
Production of a Genomic DNA Library
Authors: Quertermous Thomas 
Published: June 2004

AbstractThe describes the strategy of producing a genomic DNA library. A number of small‐scale ligations are performed using a set amount of vector and varying amounts of insert. Test ligations are transformed into bacteria (plasmid vectors) or packaged and plated on host bacteria (λ and cosmid vectors). The number of clones in the different ligations is compared, and the optimum ratio of vector to insert is indicated by the ligation with the most recombinant clones. A large‐scale ligation is then set up using this optimum ratio. This protocol employs a bacteriophage vector; however, cosmid or plasmid vectors can be used with minor modifications.
Amplification of a Bacteriophage Library
Authors: Klickstein Lloyd B. 
Published: June 2004

AbstractThis protocol may be used for genomic DNA or cDNA libraries. A freshly packaged and titered library is adsorbed to log phase plating bacteria. The mixture is then plated at high density and allowed to grow until the plaques are just subconfluent. The phage are eluted from the plate by overnight incubation with phage buffer and the library is titered and stored.
Plating and Transferring Bacteriophage Libraries
Authors: Quertermous Thomas 
Published: June 2004

AbstractThe usefulness of a recombinant phage library depends on the ability to screen a large number of phage and identify the clone that carries the DNA sequence of interest. This unit presents a protocol in which phage are allowed to multiply in host bacteria in a thin layer of agarose on regular bacterial plates. When nitrocellulose is applied to the agarose, phage particles and unpackaged DNA adsorb to the filter to produce a replica of the plate surface. If the agarose surface is not excessively wet, there will be little spreading of the phage on the filter. Subsequent treatment of the filter with sodium hydroxide destroys the phage particles and denatures the phage DNA which then binds to the nitrocellulose. Neutralization of the filters is required to maintain the integrity of the nitrocellulose. Hybridization of these filters to a DNA or RNA probe will identify the location of the phage plaque of interest, which can then be recovered from the plate.
Purification of Bacteriophage Clones
Authors: Quertermous Thomas 
Published: June 2004

AbstractCareful purification of the clone of interest away from contaminating phage is required before growth and characterization of the clone can proceed. It is common for a “purified” clone to be contaminated by a second phage, leading to confusing results and wasted time. Several rounds of purification should be performed even if the phage appears pure as early as the secondary screening stage. In this unit, phage plates are correctly oriented to the autoradiograph film, and a region that should contain the clone of interest is sampled by toothpicking each phage plaque onto secondary plates containing a lawn of host cells. Alternatively, a plug of agarose can be taken from the primary plate, placed in suspension medium, and this solution used to plate a small secondary library. Plaques on the secondary plates are transferred to nitrocellulose filters, hybridized to a 32P‐labeled probe, and an isolated positive plaque is picked, diluted in suspension medium, and regrown. This process is repeated until the desired plaque is purified.
Use of Monoclonal Antibodies for Expression Cloning
Authors: Hollenbaugh Diane , Aruffo Alejandro , Jones Bryan , Linsley Peter 
Published: June 2004

AbstractThis unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell‐surface and intracellular proteins. The first protocol in this unit describes the cloning of cDNAs encoding cell‐surface antigens. Several steps in this protocol involve transfection procedures that are described in greater detail elsewhere in this volume. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double‐stranded cDNA.
DNA Sequencing Strategies
Authors: Slatko Barton E. , Eckert Richard L. , Albright Lisa M. , Ausubel Frederick M. 
Published: June 2004

AbstractThis unit contains a general discussion of factors that should be considered before embarking on a DNA sequencing project. In general, any sequencing strategy should include plans for sequencing both strands of the DNA fragment. Complementary strand confirmation leads to higher accuracy, especially when sequencing regions where artifacts such as “compressions” are a problem. Sequencing the opposite strand is often required to obtain accurate data for such regions. The most commonly used methods for generating appropriately sized DNA fragments for dideoxy and chemical sequencing are discussed in this unit, and the biochemistry underlying these procedures, as well as how to choose between these and alternative sequencing methods, are discussed in the introduction to this chapter.
Constructing Nested Deletions for Use in DNA Sequencing
Authors: Slatko Barton , Heinrich Peter , Nixon B. Tracy , Voytas Daniel 
Published: June 2004

AbstractNested deletions useful for dideoxy DNA sequencing are a set of deletions originating at one end of a target DNA fragment and extending various lengths along the target DNA. Each successively longer deletion brings “new“ regions of the target DNA into sequencing range (about 300 bp for normal sequencing gels) of the primer site for a general discussion of nested deletions in DNA sequencing). Two protocols for generating nested subclones via enzymatic digestion are included in this unit. In the first, a set of nested deletions is generated by exonuclease III. The primary advantage of this method is that the deletion products generated from the original clone can be recircularized to generate functional plasmids and thus do not require subcloning into another vector. An alternate method utilizes Bal 31 nuclease to generate the deletions. This method requires subcloning of the deletion fragments into a separate vector for subsequent use. Both methods require the presence of unique restriction sites in the vector that are not present in the insert DNA. Bal 31 can also be used to generate nested deletions for chemical sequencing in conjunction with specialized chemical sequencing vectors.
Preparation of Templates for DNA Sequencing
Authors: Slatko Barton E. , Heinrich Peter , Nixon B. Tracy , Eckert Richard L. 
Published: June 2004

AbstractThis unit contains protocols for preparing DNA suitable for use as dideoxy sequencing templates and as material for end labeling and chemical sequencing. In all protocols, the starting material contains the recombinant molecule to be sequenced. DNA from M13mp‐derived phage is easily prepared and is currently the most reliable source of template for large‐scale dideoxy sequencing projects. Because it is occasionally necessary or convenient to use a λ‐derived phage as a source of DNA, a protocol for preparing λ phage DNA from plate lysates is provided. Two protocols for minipreps of plasmid DNA are provided, one intended for dideoxy sequencing, the other for end labeling and chemical sequencing; they differ primarily in the way in which cellular RNA is removed. Alkali denaturation of double‐stranded DNA (necessary prior to annealing) is described, and a final protocol describes the preparation of template for thermal cycle sequencing from a single phage plaque or bacterial colony.
DNA Sequencing by the Chemical Method
Authors: Eckert Richard L. 
Published: June 2004

AbstractThis procedure is written for a novice with no chemical sequencing experience. It is assumed, however, that the investigator has experience with DNA fragment isolation and DNA labeling with [32P]dNTPs. A DNA fragment labeled at only one end with 32P or 35S is divided into four aliquots and subjected to base‐specific modification reactions. Piperidine is then added to catalyze strand scission at the modified bases. Finally, the four reactions are subjected to electrophoresis on adjacent lanes of a high‐resolution denaturing polyacrylamide gel. The gel is then autoradiographed and the sequence is read from the film.
Computer Manipulation of DNA and Protein Sequences
Authors: Cherry J. Michael 
Published: June 2004

AbstractThis unit outlines a variety of methods by which DNA sequences can be manipulated by computers. Procedures for entering sequence data into the computer and assembling raw sequence data into a contiguous sequence are described first, followed by a description of methods of analyzing and manipulating sequences‐‐e.g., verifying sequences, constructing restriction maps, designing oligonucleotides, identifying protein‐coding regions, and predicting secondary structures. This unit also provides information on the large amount of software available for sequence analysis.The appendix to this unit lists some of the commercial software, shareware, and free software related to DNA sequence manipulation. The goal of this unit is to serve as a starting point for researchers interested in utilizing the tremendous sequencing resources available to the computer‐knowledgeable molecular biology laboratory.
Oligonucleotide‐Directed Mutagenesis without Phenotypic Selection
Authors: Kunkel Thomas A. 
Published: June 2004

AbstractA DNA sequence can be specifically altered by synthesizing the desired sequence change within an oligonucleotide, and then converting this into a biologically active circular DNA strand by using the oligonucleotide to prime in vitro synthesis on a single‐stranded circular template. This protocol uses a DNA template containing a small number of uracil residues in place of thymine. Use of the uracil‐containing template allows rapid and efficient recovery of mutants; in principle this same template can be applied to most of the other mutagenesis protocols in use.
Gene Synthesis: Assembly of Target Sequences Using Mutually Priming Long Oligonucleotides
Authors: Moore David D. 
Published: June 2004

AbstractThis protocol uses pairs of oligonucleotides annealed at a short duplex segment at their 3' ends as both templates and primers (mutually primed synthesis) to generate desired sequences up to 400 bp in a single step. The procedure is divided into three sections: design of the oligonucleotides, extension by mutually primed synthesis, and cloning of the extension products. The strategy for design of oligonucleotides is discussed in the commentary.
Transfection by Electroporation
Authors: Potter Huntington 
Published: June 2004

AbstractElectroporation‐‐the use of high‐voltage electric shocks to introduce DNA into cells‐‐can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. In this unit, the describes the electroporation of mammalian cells and an outlines modifications for preparation and transfection of plant protoplasts.
Overview of Genetic Reporter Systems
Authors: Kain Steven R. , Ganguly Subinay 
Published: June 2004

AbstractOne method to gauge changes in transcription is to link a presumed cis‐acting sequence(s) from a gene of interest to the coding sequence for an unrelated reporter gene. Following introduction of the chimeric reporter construct into an appropriate cell type or animal, measurement of reporter‐gene product provides an indirect estimate of the induction in gene expression directed by the regulatory sequences. Numerous in vitro and in vivo reporters genes are discussed in this overview with regard to important issues regarding the selection of a transcription reporter gene, and their applications and limitations.
Isotopic Assays for Reporter Gene Activity
Authors: Kingston Robert E. , Sheen Jen , Moore David 
Published: June 2004

AbstractThis unit describes two widely used reporter systems that are based on radioactive detection assays. The first assay uses chloramphenicol acetyltransferase (CAT) activity as a measure of the level of expression of a transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group from acetyl CoA (or any of several other acyl CoA cofactors) to chloramphenicol. In the assays described here, transfected cells are harvested and lysed, and then acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and modified derivatives of the antibiotic are separated from the starting material using either thin‐layer chromatography or phase‐extraction. The second reporter system uses a kit to perform a simple wo‐site radioimmunoassay to quantitate the amount of human growth hormone (hGH) secreted into culture medium by transfected cells. Medium is incubated with 125I‐labeled antibody specific for hGH, and immune complexes are collected by an avidin‐coated bead. The quantity of hormone is determined based on comparison with a standard curve.