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A Laboratory Guide to Genomic Sequencing - The Direct Sequencing of Native Uncloned DNA

English · Paperback / Softback

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A Safety Considerations Genomic sequencing involves a number of hazardous steps, such as high current, high voltage, radioactive and highly toxic chemicals. It is, therefore, absolutelyessen­ tial that the instructions of equipment manufacturers be followed and that particular attention is paid to the local and federal safety regulations. INTRODUCTION 9 B Introduction During the cloning of genomic DNA many of its characteristics are perma­ nently lost. It was therefore necessary to develop a new technique that would give us a closer look at a gene in its normal environment. The powerful technique of genomic sequencing, first described by Church and Gilbert (1984) now makes it possible to have a precise view of a given DNA sequence in a chromosome. This method combines the chemical DNA-sequencing procedure of Maxam and Gilbert (1980) with the detection of DNA sequences by electroblotting and indirect end-labeling by hybridization. Besides studies on the methylation state of single bases in a given gene (Nick et al. , 1986; Saluz and Jost, 1986; Saluz et al. , 1986), genomic sequencing can also be used to study specific DNA-protein interactions in vivo (Church et al. , 1985; Giniger et al. , 1985; Becker et al. , 1986; Ephrussi et al. , 1985; Martin et al. , 1986; Nick et al. , 1986; Zinn and Maniatis, 1986).

List of contents

I. Introduction.- A Safety Considerations.- B Introduction.- C The Principle of Genomic Sequencing.- II Theoretical Background.- A Basic Theoiy of Genomic Sequencing.- B Flow Diagram.- III Experimental.- 1 Isolation of Genomic DNA.- 2 Restriction Digest of Genomic DNA.- 3 Chemical Sequencing Reactions on Restricted DNA.- 4 Separation of Reaction Products on a Sequencing Gel.- 5 Electrotransfer to Nylon Membranes.- 6 Immobilization of DNA on a Nylon Membrane.- 7 Prehybridization and Hybridization of Immobilized DNA with Labeled Single-Stranded DNA Probes.- 8 Processing of the Hybridized Filters.- 9 Autoradiography and Photography.- 10 Cloning of DNA Probe in M13.- 11 Large-Scale Preparation of Cloned DNA in M13.- 12 Synthesis of Oligonucleotide Primers and Single-Stranded Labeled Probes.- 13 Purification of Labeled Single-Stranded Probes.- IV Trouble-Shooting Guide and Examples.- V Appendix.- VI Bibliography.

Product details

Authors Jost, Jost, Jean-Pierre Jost, SaLu, Saluz, Saluz, Hanspeter Saluz
Publisher Springer, Basel
 
Languages English
Product format Paperback / Softback
Released 01.01.1987
 
EAN 9783764319250
ISBN 978-3-7643-1925-0
No. of pages 164
Dimensions 171 mm x 240 mm x 15 mm
Weight 475 g
Illustrations 164 p. 31 illus.
Series Biomethods
Biomethods
Subjects Humanities, art, music > Humanities (general)
Natural sciences, medicine, IT, technology > Natural sciences (general)
Social sciences, law, business > Social sciences (general)

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