Cot Analysis Stages a Revival

A major obstacle in genome sequencing, particularly in plants, is separating the protein-encoding genes from the repeats. Researchers at University of Georgia, Athens (UGA), using sorghum as a test case, have streamlined this process using a technique popular during the 1960s and 1970s--analysis of the renaturation kinetics of DNA, familiarly known as Cot curves.1 The revived approach is called CBCS, for Cot-based cloning and sequencing.2 The term "Cot" refers to the DNA concentration (Co) multiplied by incubation time (t). As DNA double helices are separated, the resulting single-stranded molecules reassociate into double helices; the more copies present, the faster the reassociation. Repeats rejoin faster and have a lower Cot value; more complex sequences have a harder time finding each other in the mix. A Cot curve plots the fraction of reassociated DNA against the logarithm of the Cot value. CBCS has something for everyone, for it shatters and sorts genome pieces into three classes--highly repetitive, moderately repetitive, and single- or low-copy sequences. The technique can reveal genome size and complexity, and CBCS can greatly reduce the number of clones needed to sequence a genome by excluding repeats. In onion, which is 98% repeats, CBCS cuts the number of required clones from 119 million to 15 million. "It can reduce the cost of sequencing entire genomes by 50% to 95%," estimates Andrew Paterson, director of the UGA Plant Genome Mapping Laboratory. CBCS is the brainchild of Daniel Peterson, research coordinator with the Plant Genome Mapping Lab and assistant professor of plant and soil sciences at Mississippi State University. As a graduate student at Colorado State University working with Stephen Stack, Peterson performed a Cot analysis of tomato.3 Then he wrote a grant proposal to use fluorescence in situ hybridization to study sorghum chromosomes with Paterson at UGA. That study required Cot analysis. At Georgia, Peterson and associates Alexander Nagel and Scott Lee cloned the three classes of sorghum DNA. "At this point, the whole Cot cloning project was just an intellectual and technical exercise, a side project." When Peterson's sorghum chromosome studies hit a roadblock, he started spending more time on the Cot analysis. Gradually, the implications of Cot analysis and genomics began to emerge, as did the irony of using a tool invented decades before "genomics" was a word. Peterson wrote up the work, and the paper passed among the group. "The idea of CBCS was starting to evolve, but slowly. After a while, Andy asked if I would add a table showing the number of Cot clones that would have to be sequenced in order to capture the sequence complexity of the different sorghum kinetic components. I did this, and for comparison I showed the relative number of clones that would be required by shotgun sequencing to obtain similar coverage. After seeing my table, Andy recognized that we were onto something big. Up until that point we had been thinking in terms of genes, not entire genomes. I quickly found every Cot curve paper I could. We showed this to UGA professor of botany and genetics Sue Wessler and she became convinced that we had stumbled onto something pretty good," recalls Peterson. Roy Britten is excited. The inventor of Cot analysis and now adjunct professor in the department of ecology and evolutionary biology at the University of California, Irvine, Britten had helped Peterson in graduate school. "It is wonderful to find that after all these years Cot analysis could be useful again." Ricki Lewis (rlewis@nasw.org) is a contributing editor. References 1. R.J. Britten, D.E. Kohne, "Repeated sequences in DNA," Science, 161:529-40, 1968. 2. D.G. Peterson et al., "Integration of Cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery," Genome Research, 12:795-807, May 2002. 3. D.G. Peterson et al., "Characterization of the tomato (Lycopersicon esculentum) genome using in vitro and in situ DNA reassociation," Genome, 41:346-56, 1998.

Cot Analysis Stages a Revival

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