Cot analysis

C₀t analysis, also known as DNA reassociation kinetics, is a biochemical technique that measures how much repetitive DNA is in a genome.^[1] It is used to study genome structure and organization and has also been used to simplify the sequencing of genomes that contain large amounts of repetitive sequence.^[2]

Procedure

The procedure involves heating a sample of genomic DNA until it denatures into the single stranded-form, and then slowly cooling it, so the strands can pair back together. While the sample is cooling, measurements are taken of how much of the DNA is base paired at each temperature.

The amount of single and double-stranded DNA is measured by rapidly diluting the sample, which slows reassociation, and then binding the DNA to a hydroxylapatite column. The column is first washed with a low concentration of sodium phosphate buffer, which elutes the single-stranded DNA, and then with high concentrations of phosphate, which elutes the double stranded DNA. The amount of DNA in these two solutions is then measured using a spectrophotometer.

Repetitive DNA sequences renature at lower C₀t values than single-copy sequences.

Analysis

Since a sequence of single-stranded DNA needs to find its complementary strand to reform a double helix, common sequences renature more rapidly than rare sequences. Indeed, the rate at which a sequence will reassociate is proportional to the number of copies of that sequence in the DNA sample. A sample with a highly-repetitive sequence will renature rapidly, while complex sequences will renature slowly.

However, instead of simply measuring the percentage of double-stranded DNA versus time, the amount of renaturation is measured relative to a C₀t value. The C₀t value is the product of C₀ (the initial concentration of DNA), t (time in seconds), and a constant that depends on the concentration of cations in the buffer. Repetitive DNA will renature at low C₀t values, while complex and unique DNA sequences will renature at high C₀t values.

Application to genome sequencing

Main article: Cot filtration

Cot filtration is a technique that uses the principles of DNA renaturation kinetics to separate the repetitive DNA sequences that dominate many eukaryotic genomes from "gene-rich" single/low-copy sequences.^[2] This allows DNA sequencing to concentrate on the parts of the genome that are most informative and interesting, which will speed up the discovery of new genes and make the process more efficient.^[3][4]

History

It was first developed and utilized by Roy Britten and his colleagues at the Carnegie Institution of Washington in the 1960s.^[5][6] Of particular note, it was through Cot analysis that the redundant (repetitive) nature of eukaryotic genomes was first discovered.^[7] However, it wasn't until the breakthrough DNA reassociation kinetics experiments of Britten and his colleagues that it was shown that not all DNA coded for genes. In fact, their experiments demonstrated that the majority of eukaryotic genomic DNA is composed of repetitive, non-coding elements.^[1]

See also

• Cot Filtration

References

1. ^ ^{a b} Waring M, Britten RJ (1966). "Nucleotide sequence repetition: a rapidly reassociating fraction of mouse DNA". Science 154 (3750): 791–4. doi:10.1126/science.154.3750.791. PMID 5919450. 
2. ^ ^{a b} Peterson DG, Wessler SR, Paterson AH (2002). "Efficient capture of unique sequences from eukaryotic genomes". Trends Genet. 18 (11): 547–50. doi:10.1016/S0168-9525(02)02764-6. PMID 12414178. 
3. ^ LLamoureux D, Peterson DG, Li W, Fellers JP, Gill BS (2005). "The efficacy of Cot-based gene enrichment in wheat (Triticum aestivum L.)". Genome 48 (6): 1120–6. doi:10.1139/g05-080. PMID 16391681. http://article.pubs.nrc-cnrc.gc.ca/ppv/RPViewDoc?issn=0831-2796&volume=48&issue=6&startPage=1120. 
4. ^ Yuan Y, SanMiguel PJ, Bennetzen JL (2003). "High-Cot sequence analysis of the maize genome". Plant J. 34 (2): 249–55. doi:10.1046/j.1365-313X.2003.01716.x. PMID 12694599. 
5. ^ Davidson EH, Britten RJ (1973) Organization, transcription, and regulation in the animal genome. Quart. Rev. Biol. 48: 565-613.
6. ^ Britten RJ, Graham DE, Neufeld BR (1974). "Analysis of repeating DNA sequences by reassociation". Meth. Enzymol.. Methods in Enzymology 29 (0): 363–418. doi:10.1016/0076-6879(74)29033-5. ISBN 9780121818920. PMID 4850571. 
7. ^ Britten RJ, Kohne DE (1968). "Repeated sequences in DNA". Science 161 (841): 529–540. doi:10.1126/science.161.3841.529. PMID 4874239. 

External links

• Cot Analysis: An Overview Mississippi Genome Exploration Laboratory