CDNA Preparation and Characterization, Volume 303
Sherman M. Weissman
Academic Press, 1999 - Science - 575 pages
Genomic sequences, now emerging at a rapid rate, are greatly expediting certain aspects of molecular biology. However, in more complex organisms, predicting mRNA structure from genomic sequences can often be difficult. Alternative splicing, the use of alternative promoters, and orphan genes without known analogues can call present difficulties in the predictions of the structure of mRNAs or even in gene detection. Both computational and experimental methods remain useful for recognizing genes and transcript templates, even in sequenced DNA. Methods for producing full-length cDNAs are important for determining the structures of the proteins the mRNA encodes, the positions of promoters, and the considerable regulatory information for translation that may be encoded in the 5' untranslated regions of the mRNA.
Methods for studying levels of mRNA and their changes in different physiological circumstances are rapidly evolving, and the information from this area will rival the superabundance of information derived from genomic sequences. In particular, cDNAs can be prepared even from single cells, and this approach has already yielded valuable information in several areas. To the extent that reliable and reproducible information, both quantitative and qualitative, can be generated from very small numbers of cells, there are rather remarkable possibilities for complementing functional and genetic analysis of developmental patterns with descriptions of changes in mRNAs. Dense array analysis promises to be particularly valuable for the rapid expression pattern of known genes, while other methods such as gel display approaches offer the opportunity of discovering unidentified genes or for investigating species whose cDNAs or genomes have not been studied intensively.
Knowledge of mRNA structure, genomic location, and patterns of expression must be converted into information of the function of the encoded proteins. Each gene can be the subject of years of intensive study. Nevertheless, a number of methods are being developed that use cDNA to predict properties or permit the selective isolation of cDNAs encoding proteins with certain general properties such as selective isolation of cDNAs encoding proteins with certain general properties such as subcellular location. This volume presents an update of a number of approaches relevant to the areas referred to above. The technology in this field is rapidly evolving and these contributions represent a "snapshot in time" of the number of currently available and useful approaches to the problems referred to above.
The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.
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Acad agarose gel analysis anchor primer annealing arrays assay bands beads binding blots buffer cDNA clones cDNA libraries cDNA selection cDNA synthesis cells centrifuge chromosome coli concentration containing cosmid cycles database denatured differential display differentially expressed digested diluted DNA polymerase dNTPs Edited EDTA ethanol exon filter fingerprinting first-strand fragments fusion proteins gene expression Genet genomic DNA hybridization identify Incubate insert interaction isolated labeled ligation membranes method MgCl2 microcentrifuge tube mixture Molecular molecules mRNA Natl Northern blot Nucleic Acids Nucleic Acids Res oligonucleotide PCR products pellet performed plasmid plates poly(A Polymerase Chain Reaction positional cloning precipitation prepared probe Proc protocol purified reagents region restriction enzyme Resuspend reverse transcription room temperature sample screening second-strand signal solution splice step subtracted target technique template tester tion tissue total RNA transformation Tris-HCl pH TTT TTT TTT tube vector Volume washed yeast