Buch, Englisch, 164 Seiten, Format (B × H): 178 mm x 254 mm, Gewicht: 329 g
Reihe: Biomethods
The Direct Sequencing of Native Uncloned DNA
Buch, Englisch, 164 Seiten, Format (B × H): 178 mm x 254 mm, Gewicht: 329 g
Reihe: Biomethods
ISBN: 978-3-7643-1925-0
Verlag: Springer
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).
Zielgruppe
Research
Autoren/Hrsg.
Fachgebiete
Weitere Infos & Material
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.
