COT-BASED CLONING AND SEQUENCING (CBCS): A 'DIVIDE AND CONQUER' APPROACH TO GENOME EXPLORATION

¹ Mississippi Genome Exploration Laboratory, Mississippi State University, 117 Dorman Hall, Mississippi State, MS 39762, USA

² Plant Genome Mapping Laboratory, University of Georgia, CAGT Building, Athens, GA 30602, USA

Cot-Based Cloning and Sequencing (CBCS), a novel synthesis of Cot analysis, molecular cloning, and high-throughput DNA sequencing, should greatly accelerate comparative genomics research and gene discovery in organisms with repetitive genomes. In CBCS, DNA renaturation kinetics (i.e., Cot) methods are used to fractionate genomic DNA into low-copy and repetitive sequence components, each isolated kinetic component is used to construct a corresponding ‘Cot library,’ and clones are sequenced using automated DNA analyzers. If one desires to efficiently capture an organism’s sequence complexity, clones from each library can be sequenced in numbers proportional to the kinetic complexity (estimated sequence complexity) of the component from which they were derived. For repetitive eukaryotic genomes, CBCS should permit sequencing of all low-copy DNA elements at 4-20 times the efficiency of traditional whole genome shotgun sequencing. CBCS [1] allows low-copy elements to be isolated and sequenced in a manner independent of methylation patterns that vary widely between species, genes within an organism, and developmental stages, [2] is independent of gene expression patterns and thus affords access to regulatory sequences, introns, and genes that are expressed for short intervals, at low levels, and/or in response to unusual stimuli, [3] is the only sequence enrichment technique that affords an efficient means of characterizing genomic repeats as well as genes. Since it was first described, CBCS has been continually refined and improved. Such improvements have increased its usefulness as a tool in physical mapping, marker development, polymorphism discovery, and species improvement via marker-aided selection.