COT-BASED CLONING & SEQUENCING

Cot-Based Cloning and Sequencing (CBCS), a novel synthesis of Cot analysis (DNA renaturation kinetics), molecular cloning, and high-throughput DNA sequencing, is being used to accelerate comparative genomics research and gene discovery in organisms with repetitive genomes. The steps in CBCS are as follows: [1] a Cot analysis is performed for a species of interest, [2] the results of the Cot analysis are used to guide the hydroxyapatite chromatography-based fractionation of the genome into low-copy and repetitive sequence components, [3] each isolated kinetic component is used to construct a corresponding Cot library, and [4] clones from each library are sequenced in numbers proportional to the kinetic (sequence) complexity of the component from which they were derived.  For some plant and animal genomes, CBCS should permit sequencing of all low-copy DNA elements at 4-20 times the efficiency of shotgun sequencing.

Unlike methyl-filtration and PstI cloning, CBCS 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. In contrast to EST sequencing, CBCS provides access to regulatory sequences and also secures genes independently of their levels or (tissue or organ-specific) patterns of expression, increasing the likelihood of discovering key regulatory genes that are only transiently expressed.