Description
Book SynopsisA 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).
Table of ContentsI. 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.- A Suppliers of Special Items and Constructions of Commercially Unavailable Equipment.- B Determination of DNA Concentration.- VI Bibliography.
