C₀t analysis, a technique based on the principles of DNA reassociation kinetics, is a biochemical technique that measures how much repetitive DNA is in a DNA sample such as a

genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...

. It is used to study

structure and organization and has also been used to simplify the sequencing of genomes that contain large amounts of repetitive sequence.

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 Hydroxyapatite, also called hydroxylapatite (HA), is a naturally occurring mineral form of calcium apatite with the formula Ca5(PO4)3(OH), but it is usually written Ca10(PO4)6(OH)2 to denote that the crystal unit cell comprises two entities. ...

column. The column is first washed with a low concentration of

sodium phosphate Sodium phosphate is a generic term for a variety of salts of sodium (Na+) and phosphate (PO43−). Phosphate also forms families or condensed anions including di-, tri-, tetra-, and polyphosphates. Most of these salts are known in both anhyd ...

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. CoT analysis

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 Proportionality, proportion or proportional may refer to: Mathematics * Proportionality (mathematics), the property of two variables being in a multiplicative relation to a constant * Ratio, of one quantity to another, especially of a part compare ...

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

cation An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conve ...

s in the buffer. Repetitive DNA will renature at low C₀t values, while complex and unique DNA sequences will renature at high C₀t values. The fast renaturation of the repetitive DNA is because of the availability of numerous complementary sequences. Cot value = Co × t × Buffer factor Above equation is also referred as Cot equation.

Application to genome sequencing

C₀t filtration is a technique that uses the principles of DNA renaturation kinetics to separate the repetitive DNA

sequence In mathematics, a sequence is an enumerated collection of objects in which repetitions are allowed and order matters. Like a set, it contains members (also called ''elements'', or ''terms''). The number of elements (possibly infinite) is called ...

s that dominate many

eukaryotic Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bact ...

genomes In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding gen ...

from "gene-rich" single/low-copy sequences. 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.

History

It was first developed and utilized by Roy Britten and his colleagues at the

Carnegie Institution of Washington The Carnegie Institution of Washington (the organization's legal name), known also for public purposes as the Carnegie Institution for Science (CIS), is an organization in the United States established to fund and perform scientific research. Th ...

in the 1960s. Of particular note, it was through C₀t analysis that the redundant (repetitive) nature of

genomes was first discovered.{{cite journal , vauthors = Britten RJ, Kohne DE , year = 1968 , title = Repeated sequences in DNA , journal = Science , volume = 161 , issue = 841, pages = 529–540 , doi = 10.1126/science.161.3841.529 , pmid = 4874239 , bibcode = 1968Sci...161..529B 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.

References

External links

Cot Analysis: An Overview
Mississippi Genome Exploration Laboratory Molecular biology Laboratory techniques