COT FILTRATION & CBCS
What is Cot filtration?
Cot filtration (CF), a technique rooted in the principles of DNA renaturation kinetics (i.e., Cot analysis), is a means by which the repetitive DNA sequences that dominate many eukaryotic genomes can be separated from "gene-rich" single/low-copy sequences. Briefly, when sheared genomic DNA in solution is heated to near boiling temperature, the molecular forces holding complementary base pairs together are disrupted, and the two strands of each double-helix dissociate or denature. If the denatured DNA is then slowly returned to a cooler temperature, sequences will begin to reassociate (renature) with complementary strands. The temperature at which renaturation occurs can be regulated so that little or no sequence mismatch is tolerated. As predicted by the law of averages, the rate at which a sequence finds a complementary strand with which to hybridize is directly related to that sequence's iteration in the genome. In other words, those sequences that are extremely abundant (on average) find complementary strands with which to pair relatively quickly while single-copy sequences take a much longer time to find complements. In CF, genomic DNA is heat-denatured and allowed to renature to a Cot value (Cot = DNA concentration x time x a factor based on the cation concentration of the buffer) at which the majority of repetitive elements have reassociated but single and low-copy elements remain single stranded. Double-stranded, repetitive DNA is separated from single-stranded, low-copy DNA by hydroxyapatite chromatography or other means. CF allows the single/low copy sequences and the repetitive sequences of a genome to be studied independently of each other. It can also be used to fractionate highly repetitive DNA from moderately repetitive sequences or to further fractionate isolated kinetic components. CF is most accurately performed if fractionation is based upon the results of a Cot analysis.
What is Cot-Based Cloning and Sequencing (CBCS)?
Cot-Based Cloning and Sequencing (CBCS) is an invention that in which Cot-filtered DNA components are cloned and sequenced. It has become an important "reduced representation sequencing" technique and has been used to efficiently explore the gene space (and/or the repeat space) of a number of organisms. CBCS was invented by MGEL's Daniel G. Peterson and the University of Georgia's Andrew H. Paterson. Compared to shotgun sequencing using capillary-based DNA analyzers, CBCS should reduce the cost of obtaining the unique DNA sequence information from complex genomes by tens- to hundreds-of-millions of dollars. Further description of CBCS and its advantages can be found in the following sources:
| Non-Technical Description (article in Genome Technology, July 2002) | |||
| GO | CBCS Technical Summary | ||
| GO | CBCS in the news! | ||
| Additional references for Figure 4, Peterson et al. 2002b |
What now?
Recent technical advances have allowed development of DNA sequencers that can efficiently sequence megabase quantities of sheared genomic DNA without prior DNA cloning. MGEL is proud to be a partner with Pennsylvania State University's John E. Carlson in exploring the combination of Cot filtration and Roche's 454 Genome Sequencer 20 (GS20).
Other Genomics Research Topics: Bioinformatics | DNA Libraries | Cot Analysis
