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Mapping of gene transcripts by nuclease protection assays and cDNA primer extension

Calzone, Frank J. and Britten, Roy J. and Davidson, Eric H. (1987) Mapping of gene transcripts by nuclease protection assays and cDNA primer extension. In: Guide to Molecular Cloning Techniques. Methods in Enzymology. Vol.152. No.152. Academic Press , San Diego, CA, pp. 611-632. ISBN 978-0-12-182053-4. https://resolver.caltech.edu/CaltechAUTHORS:20160518-103337037

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Abstract

An important problem often faced in the molecular characterization of genes is the precise mapping of those genomic sequences transcribed into RNA. This requires identification of the genomic site initiating gene transcription, the location of genomic sequences removed from the primary gene transcript during RNA processing, and knowledge of the sequences terminating the processed gene transcript. The objective of the protocols described here is the generation of transcription maps utilizing relatively uncharacterized gene fragments. The basic approach is hybridization of a single-stranded DNA probe with cellular RNA, followed by treatment with a single-strand-specific nuclease that does not attack DNA-RNA hybrids, in order to destroy any unreacted probe sequences. Thus the probe sequences included in the hybrid duplexes are protected from nuclease digestion. The sizes of the protected probe fragments determined by gel electrophoresis correspond to the lengths of the hybridized sequence elements. Methodology for nuclease mapping of RNA transcripts with genomic DNA probes was first introduced by Berk and Sharp. We have adapted this technology for the purpose of obtaining transcription maps of new gene isolates that include 20-30 kb of genomic DNA, using probes as large as 2.5 kb. The information provided here will also be useful for less demanding mapping experiments. The use of RNA rather than DNA probes to map gene transcripts is presented in this volume.


Item Type:Book Section
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/0076-6879(87)52069-9DOIArticle
http://www.sciencedirect.com/science/article/pii/0076687987520699PublisherArticle
Additional Information:© 1987 Academic Press, Inc. Research from this laboratory was supported by NIH Grant HD-05753. F.J.C. was supported by a Senior Fellowship by the American Cancer Society, California Division.
Funders:
Funding AgencyGrant Number
NIHHD-05753
American Cancer Society, California DivisionUNSPECIFIED
Series Name:Methods in Enzymology
Issue or Number:152
Record Number:CaltechAUTHORS:20160518-103337037
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160518-103337037
Official Citation:Frank J. Calzone, Roy J. Britten, Eric H. Davidson, [66] Mapping of gene transcripts by nuclease protection assays and cDNA primer extension, Methods in Enzymology, Academic Press, 1987, Volume 152, Pages 611-632, ISSN 0076-6879, ISBN 9780121820534, http://dx.doi.org/10.1016/0076-6879(87)52069-9. (http://www.sciencedirect.com/science/article/pii/0076687987520699)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67171
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:18 May 2016 18:22
Last Modified:03 Oct 2019 10:03

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