Allan M. Maxam

[57] Sequencing end-labeled DNA with base-specific chemical cleavages

Publisher Summary This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.

Identification and location of the histone H2A and H3 genes by sequence analysis of sea urchin (S. purpuratus) DNA cloned in E. coli

A 2000 base pair (bp) DNA fragment can be excised from sea urchin (S. purpuratus) histone gene repeat units with restriction endonuclease Eco R1. This DNA, which has been cloned in a bacterial plasmid, is known to encompass two of the five histone genes. The fragment has a single endonuclease Hind III cleavage site in one of the genes and a Hae III cleavage site in the other gene. We now report the nucleotide sequences of 62 bp adjacent to the Hind III site and 42 bp adjacent to the Hae III cleavage site. The results identify the cloned DNA as histone genes, show that it codes for histone proteins H2A and H3, and locate and orient H2A and H3 genes with respect to restriction endonuclease sites in the repeat unit.