The Nucleic Acid Protocols Handbook

Front Cover
Ralph Rapley
Springer Science & Business Media, Mar 24, 2000 - Science - 478 pages
There can be no doubt that some ofthe most spectacular advances made in science over the past few decades have been in the isolation, analysis, and manipulation of nucleicacids. Thishas ledtoamuchgreaterunderstandingofmechanismsandprocesses across many fields of bioscience, such as biochemistry, microbiology, physiology, pharmacology, and the medical sciences to name a few. It has also led to the growth of the biotechnology industry, which seeks to develop and commercialize many ofthese important processes and methods. Much ofthis has come about because ofthe devel- opment of numerous molecular biology and genetic manipulation techniques. The discovery of restriction enzymes and the development of cloning vectors in the early 1970sopenedthedoortowaysofisolatingandmanipulatingnucleic acidsthathadnever been thought possible. Gene probe labeling and hybridization were developed and refined toprovidepowerfulmethodsofanalysis. These-togetherwiththedevelopment of DNA sequencing methods, protein engineering techniques, and PeR-have all continued to contribute substantially to the understandingofbiological processes at the molecular level. Theprotocols for these importantmethods are the focus ofThe Nucleic AcidProtocols Handbook, whose aim is to provide a comprehensive set oftechniques in onevolume thatwill enable the isolation, analysis, and manipulationofnucleic acids to be readily undertaken. The NucleicAcidProtocols Handbook is divided into 10 parts; within each there are approximately 10chapters. The first fourpartsfollow oneanotherlogically: nucleic acid extraction (Part I), basic separation and analysisofDNA (II), through probe design and labeling (III), and RNA analysis techniques (IV). The following three sections deal with gene libraryconstruction andscreening(V), DNA sequencing (VI), andthe polymerase chain reaction (VII).
 

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Contents

NUCLEIC ACID EXTRACTION
3
An Improved Method to Isolate Mitochondrial
23
Isolation of Fungal Nucleic Acids
37
Simultaneous RNA and DNA Extraction
53
Agarose Gel Electrophoresis of Nucleic Acids
67
Preparation of RNA Dot Blots
71
HPLC of DNA and PCR Products
105
Nick Translation and Random Hexamer Labeling of DNA
123
Cloning Gene Family Members Using Polymerase Chain
595
Construction of Synthetic Genes by Polymerase Chain Reaction
609
Inverse Polymerase Chain Reaction
625
Long Range Polymerase Chain Reaction
633
Gene Isolation by Exon Trapping
653
DNA Rescue by the Vectorette Method
667
Detection of Mutations in DNA and RNA by Chemical Cleavage
685
Detecting Point Mutations
705

Preparation of Direct EnzymeLabeled DNA Probes
145
Hybridization and Competition Hybridization of Southern Blots
163
RNA Probes for the Analysis of Gene Expression
181
Primer Extension Analysis oi mRNA
195
S1 Mapping Using SingleStranded DNA Probes
201
Characterization of RNA Using Continuous RTPCR
219
Nonradioactive Northern Blotting of RNA
239
Production of DoubleStranded cDNA for Gene Library Synthesis
261
cDNA Library Construction
289
Subtraction Hybridization cDNA Libraries
305
Cloning Polymerase Chain Reaction Products
319
Cloning Long Polymerase Chain Reaction Products
339
cDNA Library Construction for the Lambda ZAPBased Vectors
355
Antibody Screening of Bacteriophage tgt11
373
cDNA Library Screening with the Tetramethylammonium Chloride TMAC
389
Construction and Screening of Cosmid Libraries
405
YAC Library Storage and Transport
425
PhageDisplay Libraries of Murine
449
DNA Seouencmc
481
Direct cDNA Sequencing Using Sequential
499
Purification and Enzymatic Sequencing
505
Direct DNA Sequencing of Polymerase Chain Reaction Products
523
Direct Automated Cycle Sequencing
541
OneStep OneLane Chemical Sequencing of DNA
557
Primer Selection and Design for Polymerase Chain Reaction
581
The Amplification Refractory Mutation System
723
The Gel Shift Assay for the Analysis of DNAProtein Interactions _
745
The Southwestern Assay
773
Tanscrlptlonal Activation Analysis by the Chloramphenicol
793
MUTAGENESIS TRANSCRIPTION AND TRANSLATION IN VITRO
807
PrimerDirected SiteSpecific Mutagenesis
815
SiteDirected Mutagenesis Using a UracilContaining
827
SiteDirected Mutagenesis Using DoubleStranded
835
SiteDirected Mutagenesis with LAPCRTM Technology
845
Recombination and Mutagenesis
857
SiteDirected Mutagenesis and Gene Fusion
865
Transcription In Vitro Using Bacteriophage RNA Polymerases
875
In Vitro Translation of mRNA
885
ln Vitro Translation of mRNA
891
Manipulation of Baculovirus Vectors
907
Procedures for the Analysis and Purification
921
Detection and Immobilization of Proteins
935
Expression and Purification of Recombinant Proteins
947
GENE LOCALIZATION MAPPING N STu AND BIOINFORMATICS
981
Gene Mapping by FISH
991
Oligonucleotide PRINS DNA Synthesis
1011
In Situ PCR Amplification of Intracellular mRNA
1023
An Introduction to Biointormatics
1031
Index 1045
1044
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