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Chapter 23 Genomic DNA Sequencing Methods

1995; Elsevier BV; Linguagem: Inglês

10.1016/s0091-679x(08)61403-x

0091-679X

A. Favello, LaDeana Hillier, Richard K. Wilson,

Chromosomal and Genetic Variations

This chapter discusses the genomic DNA sequencing methods. The method currently in use for large-scale DNA sequence analysis of the Caenorhabditis elegans (C. elegans) genome employs a random or “shotgun” strategy. This involves using the physical map of the genome, in which, overlapping cosmid or Yeast Artificial Chromosome (YAC) clones have been ordered along with the six chromosomes. These genomic clones are fragmented by mechanical forces to generate random subclones that are sequenced and assembled by computer. The chapter discusses the preparation for approximately 700 random subclones, and the sequencing for each cosmid clone. This number of subclones provides nearly complete fivefold coverage of the cosmid, thus eliminating most of the need for library screening and primer-directed sequencing. The subclones are assembled using the xbap program. After the shotgun phase is complete, the cosmid is “finished” by closing gaps, resolving compressions, and double stranding. The final sequence is analyzed by computer in order to find the genes and regions of homology with other genomes, and finally is submitted to the public sequence databases. By systematically sequencing overlapping cosmids and portions of YACs, an entire genome can be completed.